Crown ether derivatives

ABSTRACT

The invention describes crown ether chelators, including crown ethers having the formula:

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser No. 10/634,336,filed Aug. 4, 2003 now U.S. Pat. No. 7,129,346, which is a CIP of U.S.application Ser. No. 10/026,302, filed Dec. 19, 2001 now U.S. Pat. No.6,962,992, which claims priority to U.S. Provisional Application No.60/258,266, filed Dec. 20, 2000, of which the disclosures are hereinincorporated by reference.

FIELD OF THE INVENTION

The invention relates to derivatives of crown ether chelators, includingchromophoric and fluorescent derivatives that are useful for chelatingmetal cations. Where the chelator is labeled with a fluorophore, it isan indicator useful for the detection, discrimination and quantificationof metal cations. The chelators are optionally substituted one or moretimes with a chemically reactive group or a conjugated substance, suchas a biological or nonbiological polymer, or a lipid.

BACKGROUND OF THE INVENTION

Metal ions play an important role in biological systems. Cells utilizemetal ions for a wide variety of functions, such as regulating enzymeactivity, protein structure, cellular signaling, as catalysts, astemplates for polymer formation and as regulatory elements for genetranscription. Metal ions can also have a deleterious effect whenpresent in excess of bodily requirements or capacity to excrete. A largenumber of natural and synthetic materials are known to selectively ornon-selectively bind to or chelate metal ions. Ion chelators arecommonly used in solution for in vivo control of ionic concentrationsand detoxification of excess metals, and as in vitro buffers. When boundto a fluorophore, ion chelators are typically used as optical indicatorsof ions and are useful in the analysis of cellular microenvironments ordynamic properties of proteins, membranes and nucleic acids.

Such indicators are also useful for measuring ions in extracellularspaces; in vesicles; in vascular tissue of plants and animals;biological fluids such as blood and urine; in fermentation media; inenvironmental samples such as water, soil, waste water and seawater; andin chemical reactors. Optical indicators for ions are important forqualitative and quantitative determination of ions, particularly inliving cells. Fluorescent indicators for metal cations also permit thecontinuous or intermittent optical determination of these ions in livingcells, and in solutions containing the ions.

A variety of fluorescent indicators that are useful for the detection ofbiologically relevant soluble free metal ions (such as Ca²⁺, Mg²⁺ andZn²⁺) have been described that utilize oxygen-containing anionic orpolyanionic chelators to bind to metal ions. In particular, fluorescentindicators utilizing a polycarboxylate BAPTA chelator have beenpreviously described (U.S. Pat. No. 4,603,209 to Tsien et al. (1986);U.S. Pat. No. 5,049,673 to Tsien et al. (1991); U.S. Pat. No. 4,849,362to DeMarinis et al. (1989); U.S. Pat. No. 5,453,517 to Kuhn et al.(1995); U.S. Pat. No. 5,501,980 to Malekzadeh et al. (1996); U.S. Pat.No. 5,459,276 to Kuhn et al. (1995); U.S. Pat. No. 5,501,980 toKaterinopoulos et al. (1996); U.S. Pat. No. 5,459,276 to Kuhn et al.(1995). Some fluorescent indicators selective for Li⁺, Na⁺ and K⁺ inaqueous or organic solution have also been described, based on thechemical modification of crown ethers (U.S. Pat. Nos. 5,134,232; and5,405,975; Gromov et al. Russian Chemical Bulletin (1999)48:6 p.1190-1192; Lockhart et al, J. C. S. Perkin I (1977) p 202-204).

In general a useful property for metal ion indicators is the ability todetect and/or quantify a selected metal ion in the presence of othermetal ions. Discrimination of Ca²⁺, Na⁺ and K⁺ ions in the presence ofother metal ions is particularly useful for certain biological orenvironmental samples. For most biological applications, it is essentialthat the indicators be effective in aqueous solutions. It is also usefulthat indicators for biological applications be relatively insensitive topH changes over the physiological range (pH 6-8) and sensitive to ionconcentrations in the physiological range (for sodium, a K_(d) of about5 mM to about 20 mM). It is also beneficial if the indicator absorbs andemits light in the visible spectrum where biological materials have lowintrinsic absorbance or fluorescence.

Also useful are chelators that possess a chemically reactive functionalgroup, so that the chelating group can be attached to polymers for usein remote sensing of ions or enhancing the solubility or localization ofthe optical sensor. Many chelators bind to intracellular proteins,altering the chelators metal binding properties. In addition, due totheir relatively small size, they are readily sequesterednon-selectively in intracellular vesicles, further limiting theireffectiveness. One means of circumventing these problems is to attachthe desired crown ether to a large, water-soluble polysaccharide, suchas dextran or FICOL, by means of modification of the polysaccharide toallow covalent attachment of the indicator. Dextrans and FICOLs areespecially suitable for this application, as they are low cost opticallytransparent above about 250 nm and available in multiple ranges ofmolecular weights.

Furthermore, polysaccharides and their conjugates are reasonablycompatible with most biological materials and do not interactsignificantly with intracellular components. Although fluorescentpolysaccharides have been previously described, as have indicatorconjugates of dextrans, none possess the advantageous properties of theindicator conjugates of the current invention.

The crown ether chelators of the invention show significant ability todiscriminate between metal ions under physiological conditions,particularly Ca²⁺, Na⁺ and K⁺ ions. This selectivity can be tailored bycareful selection of crown ether substituents. The compounds of theinvention are typically soluble in aqueous solutions.

The compounds of the invention that act as indicators for target ionsabsorb and emit light in the visible spectrum and possess significantutility as a means of detecting and quantifying certain metal ion levelsin living cells, biological fluids or aqueous solutions. Upon bindingthe target ion in the chelating moiety of the indicator, the opticalproperties of the attached fluorophore are generally affected in adetectable way, and this change is correlated with the presence of theion according to a defined standard. Compounds having relatively longwavelength excitation and emission bands can be used with a variety ofoptical devices and require no specialized (quartz) optics, such as arerequired by indicators that are excited or that emit at shorterwavelengths. These indicators are suitable for use in fluorescencemicroscopy, flow cytometry, fluoroscopy, or any other application thatcurrently utilize fluorescent metal ion indicators.

SUMMARY OF THE INVENTION

The present invention provides metal chelating compounds that arederivatives of crown ether compounds that bind many metal cationsincluding physiological relevant levels of metal cations such as sodium.These metal chelating compounds find utility in detecting, quantitatingand monitoring cations such as Na⁺, Li⁺, K⁺, Ca²⁺, Zn²⁺ and Rb⁺. Aparticular useful application is the binding of physiological levels ofsodium ions in living cells wherein the compounds of the presentinvention provide for the detection, quantitation and monitoring of theintracellular sodium ions.

The metal chelating compounds of the present invention are derivativesof crown ether compounds and have the following formula:

wherein the compound contains at least one oxygen atom and preferablythree to five oxygen atoms in the crown of the compound. The oxygenatoms are preferably separated by —(CH₂)₂—. Crown ether compounds thatcontain an oxygen and nitrogen atom ortho to the benzo moiety findparticular use in binding sodium ions and in generating a detectablesignal when bound by an -L-DYE moiety at one of the benzo substitutents.

Thus, P and Q are independently O, S or NR³, wherein each R³ isindependently H or C₁-C₆ alkyl. Typically P and Q are O.

More specifically, Y is O, S, NR⁴ or is absent. R⁴ is selected from thegroup consisting of H, -L-R_(x), -L-S_(c), -L-DYE, C₁-C₁₈ alkyl, aryland heteroaryl ring system, which alkyl or ring system is optionallysubstituted by halogen, azido, nitro, nitroso, amino, C₁-C₆ alkylamino,C₂-C₁₂ dialkylamino, cyano, -L-R_(x), -L-S_(c), -L-DYE, C₁-C₆ alkyl orC₁-C₆ alkoxy that is itself optionally substituted by halogen, amino,hydroxy, —(SO₂)—R¹⁵, —(SO₂)—O—R¹⁵, —(C═OR¹⁵, —(C═O)—O—R¹⁶, or—(C═O)—NR¹⁷R¹⁸. R¹⁵ is selected from the group consisting of H, C₁-C₆alkyl, -L-R_(x), -L-S_(c) and -L-DYE and R¹⁶ is selected from the groupconsisting of H, C₁-C₆ alkyl, benzyl, a biologically compatibleesterifying group, a biologically compatible salt, -L-R_(x), -L-S_(c)and -L-DYE. R¹⁷ and R¹⁸ are independently selected from the groupconsisting of H, C₁-C₆ alkyl, C₁-C₆ carboxyalkyl, alpha-acyloxyalkyl,trialkylsilyl, a biologically compatible salt, -L-R_(x), -L-S_(c) and-L-DYE; or R¹⁷ and R¹⁸ taken in combination form a 5- or 6-memberedaliphatic ring that optionally incorporates an oxygen atom;

R_(x) is a reactive group that is capable of forming a covalent bondwith another substance containing an appropriate reactive group to forma conjugated substance (S_(c)). Particularly useful reactive groups ofthe metal chelating compounds include carboxylic acid and activatedesters of carboxylic acid for labeling amines and alcohols ofbiomolecules.

Conjugated substances are intended to mean any biomolecule ornon-biomolecule that contains a moiety capable of forming a covalentlinkage with another moiety or is modified to contain such a reactivegroup. Particularly useful conjugated substances include proteins,peptides and non-biomolecule polymers. The covalent linkage (L) can be asingle covalent bond or a series of stable bonds containing 1-20non-hydrogen atoms including P, C, N, O and S.

E¹, E², and E³ are independently —(CR⁵ ₂)_(n)—, —(C(O)CH₂)_(n)—, —(CR⁵₂)_(n)O(CR⁵ ₂)_(n)— or E² is absent, where n=2, 3 or 4, and each R⁵ isindependently H or CH₃, or two R⁵ moieties on adjacent carbons of one ormore of E¹, E² or E³, when taken in combination, form a 5- or 6-memberedaliphatic ring.

R¹ is selected from the group consisting of -L-R_(x), -L-S_(c), -L-DYE,C₁-C₁₈ alkyl and C₇-C₁₈ arylalkyl, each of which is optionallysubstituted by halogen, azido, nitro, nitroso, amino, hydroxy, cyano, anaryl or heteroaryl ring system, —(SO₂)—R¹⁵, —(SO₂)—O—R¹⁵, —(C═O)—R¹⁵,—(C═O)—O—R¹⁶, —(C═O)—NR¹⁷R¹⁸, C₁-C₆ alkylamino, C₂-C₁₂ dialkylamino,C₁-C₆ alkyl or C₁-C₆ alkoxy and each of which are optionally furthersubstituted by halogen, amino (—NR¹⁷R¹⁸), hydroxy, —(SO₂)—R¹⁵,—(SO₂)—O—R¹⁵, —(C═O)—R¹⁵, —(C═O)—O—R¹⁶ or —(C═O)—NR¹⁷R¹⁸.

A particularly preferred R¹ is represented by methyl or ethyl that issubstituted by —(C═O)—O—R¹⁶ or —(C═O)—R¹⁵ wherein R¹⁶ or R¹⁵ is a methylgroup. These particular substitutents appear to play a role instabilizing the metal ion in the chelating ring of the presentcompounds.

R¹⁹ and R²⁰ are independently selected from the group consisting of H,halogen, azido, nitro, nitroso, amino, cyano, -L-R_(x), -L-S_(c),-L-DYE, C₁-C₆ alkyl and C₁-C₆ alkoxy, each of which is itself optionallysubstituted by halogen, amino, hydroxy, —(SO₂)—R¹⁵, —(SO₂)—O—R¹⁵,—(C═O)—R¹⁵, —(C═O)—O—R¹⁵, or —(C═O)—NR¹⁷R¹⁸. Alternatively, R¹⁹ and R²⁰taken in combination form a fused six-membered benzo moiety that isoptionally substituted by halogen, azido, nitro, nitroso, amino, cyano,-L-R_(x), -L-S_(c), -L-DYE, C₁-C₆ alkyl or C₁-C₆ alkoxy, each of whichis itself optionally substituted by halogen, amino, hydroxy, —(SO₂)—R¹⁵,—(SO₂)—O—R¹⁵,—(C═O)—R¹⁵, —(C═O)—O—R¹⁶, or —(C═O)—NR¹⁷R¹⁸.

R⁷, R⁸, R⁹ and R¹⁰ are independently selected from the group consistingof H, halogen, azido, nitro, nitroso, amino, cyano, -L-R_(x), -L-S_(c),-L-DYE, C₁-C₆ alkyl or C₁-C₆ alkoxy, each of which is itself optionallysubstituted by halogen, amino, hydroxy, —(SO₂)—R¹⁵, —(SO₂)—O—R¹⁵,—(C═O)—R¹⁵, —(C═O)—O—R¹⁶, or —(C═O)—NR¹⁷R¹⁸. Alternatively, any twoadjacent substituents R⁷—R¹⁰, taken in combination, form a fusedsix-membered benzo moiety, which is optionally substituted by halogen,azido, nitro, nitroso, amino, cyano, -L-R_(x), -L-S_(c), -L-DYE, C₁-C₆alkyl or C₁-C₆ alkoxy, each of which is optionally substituted byhalogen, amino, hydroxy, —(C═O)—R¹⁵, —(C═O)—O—R¹⁶, or —(C═O)—NR¹⁷R¹⁸. Inaddition, any two adjacent substituents R⁷—R¹⁰, or R¹⁹ and R²⁰, taken incombination with each other, form a fused DYE wherein the dye shares thebenzo moiety of the present compounds.

A particularly useful compound of the present invention is when R⁸ or R⁹is represented by a -L-DYE or R⁸ and R⁹ taken in combination form afused DYE. In addition, when a lipiphilic group such as an AM estersubstitutes the DYE moiety the present compounds find use in binding invivo metal cations. This is a particularly useful aspect of the presentinvention wherein certain embodiments of the compounds, such ascompounds containing only one nitrogen atom and one benzo moiety, entercells and bind target ions with better results compared to similarcompounds.

The compounds of the present invention are useful for bindingphysiological relevant levels of cations, particularly sodium ions. Whenthe present compounds comprise a DYE moiety the compounds find use tomonitor, detect and quantitate such cations. Furthermore, compoundscomprising a lipophilic group such as an AM or acetate ester groupprovide compounds that are cell permeable but are well retained in thecell after the lipophilic group is cleaved by nonspecific esterasesresulting in a charged molecule.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Shows the Na⁺-dependent fluorescence excitation spectra ofCompound 51 in a series of solutions containing 0 to 1000 mM free Na⁺,with fluorescence emission monitored at 510 nm (as described in Example71).

FIG. 2: Shows the Na⁺-dependent fluorescence emission spectra ofCompound 22 in a series of solutions containing 0 to 500 mM free Na⁺,with excitation at 488 nm (as described in Example 71).

FIG. 3: Shows a comparison of the intracellular sodium response ofSODIUM GREEN tetraacetate indicator (Molecular Probes, Inc.) or Compound25. At every intracellular Na⁺ concentration, Compound 25 demonstratesstronger fluorescence intensity than SODIUM GREEN sodium indicator atthe same concentration (as described in Example 72).

FIG. 4: Shows the Na+ (FIG. 4A) and K+ (FIG. 4B)-dependent fluorescentemission spectra of Compound 86 in a series of solutions containing 0 to1000 mM free Na+ and 0 to 5M of free K+ ions, with excitation at 488 nm.See, Example 71.

FIG. 5: Shows intracellular detection of Na+ ions with Compound 87. See,Example 72.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific compositionsor process steps, as such may vary. It must be noted that, as used inthis specification and the appended claims, the singular form “a”, “an”and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a fusion protein” includes aplurality of proteins and reference to “a fluorescent compound” includesa plurality of compounds and the like.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention is related. The following terms aredefined for purposes of the invention as described herein.

The term “affinity” as used herein refers to the strength of the bindinginteraction of two molecules, such as a metal chelating compound and ametal ion or a positively charged moiety and a negatively chargedmoiety.

The term “alkyl” as used herein refers to a straight, branched or cyclichydrocarbon chain fragment containing between about one and about twentyfive carbon atoms (e.g. methyl, ethyl and the like). Straight, branchedor cyclic hydrocarbon chains having eight or fewer carbon atoms willalso be referred to herein as “lower alkyl”. In addition, the term“alkyl” as used herein further includes one or more substitutions at oneor more carbon atoms of the hydrocarbon chain fragment. Suchsubstitutions include, but are not limited to: aryl; heteroaryl;halogen; alkoxy; amine (—NR′R″); carboxy and thio.

The term “amino” or “amine group” refers to the group —NR′R″ (or NR′R″)where R, R′ and R″ are independently selected from the group consistingof hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted aryl alkyl, heteroaryl, and substituted heteroaryl. Asubstituted amine being an amine group wherein R′ or R″ is other thanhydrogen. In a primary amino group, both R′ and R″ are hydrogen, whereasin a secondary amino group, either, but not both, R′ or R″ is hydrogen.In addition, the terms “amine” and “amino” can include protonated andquaternized versions of nitrogen, comprising the group —NR′R″ and itsbiologically compatible anionic counterions.

The term “aryl” as used herein refers to cyclic aromatic carbon chainhaving twenty or fewer carbon atoms, e.g., phenyl, naphthyl, biphenyl,and anthracenyl. One or more carbon atoms of the aryl group may also besubstituted with, e.g., alkyl; aryl; heteroaryl; a halogen; nitro;cyano; hydroxyl, alkoxyl or aryloxyl; thio or mercapto, alkyl-, orarylthio; amino, alkylamino, arylamino, dialkyl-, diaryl-, orarylalkylamino; aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl,dialkylaminocarbonyl, diarylaminocarbonyl, or arylalkylaminocarbonyl;carboxyl, or alkyl- or aryloxycarbonyl; aldehyde; aryl- oralkylcarbonyl; iminyl, or aryl- or alkyliminyl; sulfo; alkyl- oralkylcarbonyl; iminyl, or aryl- or alkyliminyl; sulfo; alkyl- orarylsufonyl; hydroximinyl, or aryl- or alkoximinyl. In addition, two ormore alkyl or heteroalkyl substituents of an aryl group may be combinedto form fused aryl-alkyl or arylheteroalkyl ring systems (e.g.,tetrahydronaphthyl). Substituents including heterocyclic groups (e.g.,heteroaryloxy, and heteroaralkylthio) are defined by analogy to theabove-described terms.

The term “arylalkyl” as used herein refers to an aryl group that isjoined to a parent structure by an alkyl group as described above, e.g.,benzyl, α-methylbenzyl, phenethyl, and the like.

The term “attachment site” as used herein refers to a site on a moietyor a molecule, e. g. a metal chelating compound, a fluorescent dye, apeptide or a protein, to which is covalently attached, or capable ofbeing covalently attached, to a linker or another moiety.

The term “aqueous solution” as used herein refers to a solution that ispredominantly water and retains the solution characteristics of water.Where the aqueous solution contains solvents in addition to water, wateris typically the predominant solvent.

The term “cell permeable” as used herein refers to compounds of thepresent invention that are able to cross the cell membrane of livecells. Lipophilic groups that are covalently attached to the presentcompounds, typically on the DYE moiety, facilitate this permeability andlive cell entry. Once inside the cells, the lipophilic groups arehydrolyzed resulting in charged molecules that are well retained inliving cells. Particularly useful lipophilic groups includeacetoxymethyl (AM) ester and acetate esters wherein once inside thecalls the groups are cleaved by nonspecific esterases resulting incharged molecules.

The term “complex” as used herein refers to the association of two ormore molecules, usually by non-covalent bonding.

The term “detectable response” as used herein refers to a change in oran occurrence of, a signal that is directly or indirectly detectableeither by observation or by instrumentation and the presence ormagnitude of which is a function of the presence of a target metal ionin the test sample. Typically, the detectable response is an opticalresponse resulting in a change in the wavelength distribution patternsor intensity of absorbance or fluorescence or a change in light scatter,fluorescence quantum yield, fluorescence lifetime, fluorescencepolarization, a shift in excitation or emission wavelength or acombination of the above parameters. The detectable change in a givenspectral property is generally an increase or a decrease. However,spectral changes that result in an enhancement of fluorescence intensityand/or a shift in the wavelength of fluorescence emission or excitationare also useful. The change in fluorescence on ion binding is usuallydue to conformational or electronic changes in the indicator that mayoccur in either the excited or ground state of the fluorophore, due tochanges in electron density at the ion binding site, due to quenching offluorescence by the bound target metal ion, or due to any combination ofthese or other effects. Alternatively, the detectable response is anoccurrence of a signal wherein the fluorophore is inherently fluorescentand does not produce a change in signal upon binding to a metal ion orbiological compound.

The term “DYE” as used herein refers to a reporter molecule that isinherently fluorescent or demonstrates a change in fluorescence uponbinding to a biological compound or metal ion, i.e., fluorogenic.Numerous fluorophores are known to those skilled in the art and include,but are not limited to, coumarin, acridine, furan, indole,borapolyazaindacene, cyanine, benzofuran, quinazolinone, benzazole,oxazine and xanthenes including fluoroscein, rhodamine, rosamine andrhodol, as well as other fluorophores described in RICHARD P. HAUGLAND,MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH PRODUCTS(9^(th) edition, CD-ROM, 2002). The DYE moiety may be substituted bysubstituents that enhance solubility, live cell permeability and alterspectra absorption and emission.

The term “heteroaryl” or “heteroaromatic ring system” as used hereinrefers to a 5- or 6-membered unsaturated ring that is optionally fusedto an additional six-membered aromatic ring(s), or is fused to one 5- or6-membered unsaturated ring containing one or more heteroatoms, and isoptionally substituted as defined below. Each heteroaromatic ringcontains at least 1 and as many as 3 heteroatoms that are selected fromthe group consisting of O, N or S in any combination. Specific examplesof a heteroaryl ring system include, but are not limited to, substitutedor unsubstituted derivatives of 2- or 3-furanyl; 2- or 3-thienyl; N—, 2-or 3-pyrrolyl; 2- or 3-benzofuranyl; 2- or 3-benzothienyl; N—, 2- or3-indolyl; 2-, 3- or 4-pyridyl; 2-, 3- or 4-quinolyl; 1-, 3-, or4-isoquinolyl; 2-, 4-, or 5-(1,3-oxazolyl); 2-benzoxazolyl; 2-, 4-, or5-(1,3-thiazolyl); 2-benzothiazolyl; 3-, 4-, or 5-isoxazolyl; N—, 2-, or4-imidazolyl; N—, or 2-benzimidazolyl; 1- or 2-naphthofuranyl; 1- or2-naphthothienyl; N—, 2- or 3-benzindolyl; 2-, 3-, or 4-benzoquinolyl;1-, 2-, 3-, or 4-acridinyl. Preferably the heteroaryl substituent issubstituted or unsubstituted 4-pyridyl, 2-thienyl, 2-pyrrolyl,2-indolyl, 2-oxazolyl, 2-benzothiazolyl or 2-benzoxazolyl. Morepreferably, the heteroaryl substituent is 2-thienyl or 2-pyrrolyl.

The term “kit” as used refers to a packaged set of related components,typically one or more compounds or compositions.

The term “Linker” or “L” as used herein refers to a single covalent bondor a series of stable covalent bonds incorporating 1-20 nonhydrogenatoms selected from the group consisting of C, N, O, S and P thatcovalently attach the crown ether metal chelating compounds to anothermoiety such as a chemically reactive group, a DYE or a conjugatedsubstance including biological and non-biological substances. A“cleavable linker” is a linker that has one or more covalent bonds thatmay be broken by the result of a reaction or condition. For example, anester in a molecule is a linker that may be cleaved by a reagent, e.g.sodium hydroxide, resulting in a carboxylate-containing fragment and ahydroxyl-containing product,

The term “metal chelator” or “metal chelating compound” as used hereinrefers to a chemical compound that combines with a metal ion to form achelate ring structure.

The term “metal ion” or “target metal ion” as used herein refers to anymetal cation that is capable of being chelated by the present crownether chelate compounds. Typically, these metal ions are physiologicaland or nutritional relevant metal ion such as Na⁺, K⁺, Zn²⁺ and Ca²⁺.The term metal ion used herein also refers to the metal ions Li⁺ andRb⁺.

The terms “protein” and “polypeptide” are used herein in a generic senseto include polymers of amino acid residues of any length. The term“peptide” is used herein to refer to polypeptides having less than 250amino acid residues, typically less than 100 amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidues are an artificial chemical analogue of a correspondingnaturally occurring amino acid, as well as to naturally occurring aminoacid polymers.

The term “Rx” or “reactive group” as used herein refers to a group thatis capable of reacting with another chemical group to form a covalentbond, i.e. is covalently reactive under suitable reaction conditions,and generally represents a point of attachment for another substance.The reactive group is a moiety, such as carboxylic acid or succinimidylester, on the compounds of the present invention that is capable ofchemically reacting with a functional group on a different compound toform a covalent linkage. Reactive groups generally include nucleophiles,electrophiles and photoactivatable groups.

The term “reporter molecule” as used herein refers to a chemical moietythat when covalently attached to the present crown ether compound iscapable of generating a detectable response. Typically the reportermolecule is a DYE moiety.

The term “sample” as used herein refers to any material that may containtarget metal ions, as defined above. Typically, the sample is a livecell or a biological fluid that comprises endogenous host cell proteins.Alternatively, the sample may be a buffer solution or an environmentalsample containing target metal ions. The sample may be in an aqueoussolution, a viable cell culture or immobilized on a solid or semi solidsurface such as a polyacrylamide gel, membrane blot or on a microarray.

II. Compositions and Methods of Use

A. Components of the Crown Ether Chelate Compounds

The present invention provides derivatives of crown ether compounds thatbind a wide range of metal cations including physiological relevantlevels of metal cations such as sodium. These metal chelating compoundscomprise a crown ether moiety, at least one benzo moiety, substituentswell known in the art including linkers, chemically reactive groups andDYE moieties that function as reporter groups. The crown either moietycontains at least four heteroatoms, one of which is required to be anitrogen atom and is located ortho to the benzo moiety. The remainingheteratoms may be selected from the group consisting of nitrogen, oxygenand sulfur and are selected based on their ability to bind differentmetal cations with different affinity. Typically, in addition to thenitrogen atom there is also an oxygen atom ortho to the benzo moietythat facilitates binding of target metal ions. Furthermore, thesubstituents on the nitrogen atom are further used to after the affinityfor particular metal ions under different environmental conditions.

The present compounds find utility in binding target metal ions in asample. The sample includes live cells or a biological fluid thatcomprises endogenous host cell proteins, buffer solutions andenvironmental samples. Therefore, when the present crown ether compoundscomprise a DYE moiety they find utility in quantitating, monitoring anddetecting target metal ions. Typically, the DYE moiety is directlyattached to the benzo moiety or two of the benzo substituents when takenin combination form a fused DYE moiety. Detection of target metal ionscan also be accomplished in live cells wherein the DYE moiety comprisesa lipophilic group such as an AM or acetate ester that allows for entryacross the live cell membrane. Once inside the cells nonspecificesterases cleave the AM or acetate ester resulting a charged moleculethat is well retained in the cell. These present compounds areparticularly useful for binding physiological relevant levels of sodium,potassium or calcium cations.

1. Chelating Moiety

The compounds of the invention are represented by the following twoformulas:

The heteroatom Y is O, S, NR⁴ or is absent where R⁴ is H, a C₁-C₁₈alkyl, or an aryl or heteroaryl ring system. The R⁴ alkyl or ring systemsubstituent is optionally substituted one or more times by halogen,azido, nitro, nitroso, amino, alkylamino having 1-6 carbons,dialkylamino having 2-12 carbons, cyano, or R⁴ is substituted one ormore times by a C₁-C₆ alkyl or C₁-C₆ alkoxy that is itself optionallysubstituted one or more times by halogen, amino, hydroxy, —(SO₂)—R¹⁵,—(SO₂)—O—R¹⁵, —(C═O)—R¹⁵, —(C═O)—O—R¹⁶, or —(C═O)—NR¹⁷R¹⁸.Alternatively, R⁴ is, or is substituted by -L-R_(x), -L-S_(c), or-L-DYE.

In one aspect of the invention Y is O and in another aspect Y is absentprovided that E² is also absent.

R¹⁵ is independently H or C₁-C₆ alkyl. Each R¹⁶ is H, a C₁-C₆ alkyl, abenzyl, or forms an ester (e.g., R¹⁶ is an alpha-acyloxyalkyl, atrialkylsilyl, or any other biologically compatible esterifying group).Additionally, any R¹⁶ is a biologically compatible salt. R¹⁷ and R¹⁸ areindependently H or C₁-C₆ alkyl or C₁-C₆carboxyalkyl, or analpha-acyloxyalkyl, trialkylsilyl, or any other biologically compatibleesterifying group, or a biologically compatible salt. Alternatively, R¹⁷and R¹⁸ when taken in combination form a 5- or 6-membered aliphatic ringthat optionally incorporates an oxygen atom. In addition, one or more ofa R¹⁵, R¹⁸, R¹⁷, or R¹⁸ is permitted to be -L-R_(x), -L-S_(c), or-L-DYE.

Each L is independently a covalent linkage. Each R_(x) is independentlya chemically reactive group. Each S_(c) is independently a conjugatedsubstance. Each DYE is independently a reporter molecule that is achromophore that maximally absorbs light at a wavelength greater than320 nm.

The heteroatoms P and Q are independently selected from O, S, or NR³,where each R³ is independently H or an alkyl having 1-6 carbons. In oneaspect of the invention P and Q are both O.

In another aspect of the invention, P and Q are O, and Y is NR⁴.Typically, P, Q, and Y are each O or Y is absent and P and Q are O.Careful selection of the nature of the P, Q, and Y heteroatoms permitsthe moderation of the selectivity and binding affinity of the resultingcrown ether compound.

E¹, E², and E³ each independently have the formula —(CR⁵ ₂)_(n)—, or—[C(O)CH₂]_(n)—, —(CR⁵ ₂)_(n)O(CR⁵ ₂)_(n)— or E² is absent where n is 2,3 or 4. Each R⁵ is independently H or methyl, or the R⁵ moieties onadjacent carbon atoms of each chain, when taken in combination, forms a5- or 6-membered aliphatic ring. For a given E moiety, each R⁵ istypically H and n is 2. Where n is 2 for each E moiety, the resultingcompound is known as a 15-crown-5 crown ether, having 15 atoms in thechelating ring itself, of which 5 are heteroatoms.

Alternatively, E2 is absent or a E moiety is —(CR⁵ ₂)_(n)O(CR⁵ ₂)_(n)—resulting in a chelating ring with a different number of total atoms andpossible heteroatoms such as oxygen.

In one aspect of the invention E¹, E², and E³ are each —(CH₂)₂— and Y, Pand Q are each oxygen. In another aspect, E² is absent and P and Q areoxygen wherein Y is absent and E¹ and E³ are each —CH₂)₂—. In yetanother aspect of the invention at least one of E¹, E², or E³ is —(CR⁵₂)_(n)O(CR⁵ ₂)_(n)— wherein Y, P and Q are each oxygen and the remainingE moieties are —(CH₂)₂—.

Formula (I) contains the amine substituents R¹ and R², however Formula(II) contains only R¹, the second nitrogen having been replaced by adivalent oxygen atom. Thus, it is understood that while the aminesubstitutents, R1 and R2, are referred to in plural only one is intendedfor compounds represented by formula (II). These amine substituents areindependently H, C₁-C₁₈ alkyl, or C₇-C₁₈ arylalkyl. Where R¹ or R² isalkyl or arylalkyl, it is optionally substituted one or more times byhalogen, azido, nitro, nitroso, amino, hydroxy, C₁-C₆ alkylamino, C₂-C₁₂dialkylamino, cyano, or by an aryl or heteroaryl ring system, or by—(SO₂)—R¹⁵, —(SO₂)—O—R¹⁵, —(C═O)—R¹⁵, —(C═O)—O—R¹⁶, or —(C═O)—NR¹⁷R¹⁸;or by a C₁-C₆ alkyl or C₁-C₆ alkoxy that is itself optionallysubstituted one or more times by halogen, amino, hydroxy, —(SO₂)—R¹⁶,—(SO₂)—R—R¹⁵, —(C═O)—R¹⁵, —(C═O)—O—R¹⁶, or —(C═O)—NR¹⁷R¹⁸.Alternatively, R¹ and R² are optionally -L-R_(x), -L-S_(c), or -L-DYE.

Where the R¹ and R² substituents are not -L-R_(x), -L-S_(c), or -L-DYE,they are typically both a lower alkyl that is substituted one or moretimes by carboxylic acids, by carboxylic acid esters, by carboxylic acidamides, or by cyano. Where R¹ and R² incorporate carboxylic acid estersthey are typically not cleaved by esterase but instead function tostabilize the bound metal ion in the chelate ring. Thus R¹, and R² whenpresent, are typically a methyl or ethyl that is substituted by acarboxylic acid containing group such as —(C═O)—R¹⁵ or —(C═O)—O—R¹⁶wherein R¹⁵ and R¹⁶ are each a methyl or ethyl. Selection of the precisenature of R¹ and R² can greatly affect the binding selectivity andaffinity of the resulting compound for a target ion (see, Table 1 and2).

The nature of the R¹ and R² substituents in a large part determines theresponse of the indicators to particular target metal ions. For example,where the crown ether derivatives have the formula

where R¹ and R² are each methoxycarbonylmethyl selectively bind sodiumions with a dissociation constant (K_(d)) of approximately 20-100 mM,and are relatively insensitive to the presence of potassium ions. Inparticular, the sodium ion K_(d) values typically rise less than about10% when measured in the presence of 100 mM potassium ion.

The Formula (II) substituents R¹⁹ and R²⁰ are represented by H, halogen,azido, nitro, nitroso, amino, cyano, -L-R_(x), -L-S_(c), -L-DYE, C₁-C₆alkyl and C₁-C₆ alkoxy, each of which is itself optionally substitutedby halogen, amino, hydroxy, —(SO₂)—R¹⁵, —(SO₂)—O—R¹⁵, —(C═O)—R¹⁵,—(C═O)—O—R¹⁶, or —(C═O)—NR¹⁷R¹⁸. Alternatively, R¹⁹ and R²⁰ taken incombination form a fused six-membered benzo moiety that is optionallysubstituted by halogen, azido, nitro, nitroso, amino, cyano, -L-R_(x),-L-S_(c), -L-DYE, C₁-C₆ alkyl or C₁-C₆ alkoxy, each of which is itselfoptionally substituted by halogen, amino, hydroxy, —(SO₂)—R¹⁵,—(SO₂)—O—R¹⁵, —(C═O)—R¹⁵, —(C═O)—O—R¹⁶, or —(C═O)—NR¹⁷R¹⁸. In one aspectof the invention, R¹⁹ and R²⁰ are both hydrogen. In another aspect, R¹⁹and R²⁰ form a benzo moiety that is optionally substituted resulting ina crown ether chelate compound with two benzo moieties wherein only onebenzo moiety has a nitrogen atom that is ortho to the benzo moiety.

The benzo substituents R⁷—R¹⁰, and R¹¹—R¹⁴ when present, areindependently H, halogen, azido, nitro, nitroso, amino, cyano; or-L-R_(x), -L-S_(c), or -L-DYE; or C₁-C₆ alkyl or C₁-C₆ alkoxy that isitself optionally substituted one or more times by halogen, amino,hydroxy, —(SO₂)—R¹⁵, —(SO₂)—O—R¹⁶, —(C═O)—R¹⁵, —(C═O)—O—R¹⁶, or—(C═O)—NR¹⁷R¹⁸.

Alternatively, two adjacent substituents of R⁷—R¹⁴, when taken incombination, form a fused six-membered benzo moiety, which is optionallysubstituted one or more times by a halogen, azido, nitro, nitroso,amino, cyano, -L-R_(x), -L-S_(c), or -L-DYE; or by a C₁-C₆ alkyl orC₁-C₆ alkoxy, which is Itself optionally substituted one or more timesby halogen, amino, hydroxy, —(SO₂)—R¹⁵, —(SO₂)—O—R¹⁵, —(C═O)—R¹⁵,—(C═O)—O—R¹⁶, or —(C═O)—NR¹⁷R¹⁸.

Furthermore, the present compounds comprise a fused DYE wherein twoadjacent substituents of R⁷—R¹⁴, when taken in combination with eachother, and with the aromatic ring they are bound to, form a fused DYE.In this way the benzo moiety of the present compounds is also part of aDYE moiety.

The compounds of the invention represented by Formula (I) aresubstituted by at least one -L-DYE, -L-R_(x) or -L-S_(c) at one or moreof R¹, R², R⁴, and R⁷—R¹⁴; or two of R⁷—R¹⁴, taken in combination, forma fused DYE. In one embodiment, the compounds of the invention aresubstituted by exactly one -L-DYE moiety, which is bound at R¹, R², anR⁴, or one of R⁷—R¹⁴, or is a fused DYE moiety at two adjacentsubstituents of R⁷—R¹⁴. The DYE moiety is typically bound at one ofR⁷—R¹¹, preferably at R⁹ or R⁸, or is bound at R⁴ where Y is NR⁴. In oneembodiment, compounds that are substituted by exactly one -L-DYE moietyare optionally further substituted by -L-R_(x) or -L-S_(c), typically atR¹, R², R⁴, or one of R⁷—R¹⁴.

In another embodiment, the compound of the invention is substituted byexactly two DYE moieties, which may be the same or different, and may bebound by a covalent linkage L or fused to the crown ether chelate. Inone embodiment of the invention, a first -L-DYE moiety is bound at oneof R⁷—R¹⁰, while the second -L-DYE moiety is bound at one of R¹¹—R¹⁴.Typically, the first -L-DYE moiety is bound at R⁹, while the second-L-DYE moiety is bound at R¹², or a DYE moiety is fused at R⁸ and R⁹ andadditionally at R¹² and R¹³.

2. Linkers of the Crown Ether Chelate Compounds

The crown ether chelate compounds of the present invention typicallycomprise a linker that is used to covalently attach a DYE moiety,conjugated substance or reactive group to the compound. When present,the linker is a single covalent bond or a series of stable bonds. Thus,the reporter molecule, conjugated substance or reactive group may bedirectly attached (where Linker is a single bond) to the crown etherchelate or attached through a series of stable bonds. When the linker isa series of stable covalent bonds the linker typically incorporates 1-20nonhydrogen atoms selected from the group consisting of C, N, O, S andP. In addition, the covalent linkage can incorporates a platinum atom,such as described in U.S. Pat. No. 5,714,327. When the linker is not asingle covalent bond, the linker may be any combination of stablechemical bonds, optionally including, single, double, triple or aromaticcarbon-carbon bonds, as well as carbon-nitrogen bonds, nitrogen-nitrogenbonds, carbon-oxygen bonds, sulfur-sulfur bonds, carbon-sulfur bonds,phosphorus-oxygen bonds, phosphorus-nitrogen bonds, andnitrogen-platinum bonds. Typically the linker incorporates less than 15nonhydrogen atoms and are composed of any combination of ether,thioether, thiourea, amine, ester, carboxamide, sulfonamide, hydrazidebonds and aromatic or heteroaromatic bonds. Typically the linker is asingle covalent bond or a combination of single carbon-carbon bonds andcarboxamide, sulfonamide or thioether bonds. The bonds of the linkertypically result in the following moieties that can be found in thelinker ether, thioether, carboxamide, thiourea, sulfonamide, urea,urethane, hydrazine, alkyl, aryl, heteroaryl, alkoxy, cycloalkyl andamine moieties. Examples of L include substituted or unsubstitutedpolymethylene, arylene, alkylarylene, arylenealkyl, or arylthio.

In one embodiment, L contains 1-6 carbon atoms; in another, L comprisesa thioether linkage. In another embodiment, L is or incorporates theformula —(CH₂)_(d)(CONH(CH₂)_(e))_(z)— or—O(CH₂)_(d)(CONH(CH₂)_(e))_(z)—, where d is an integer from 0-5, e is aninteger from 1-5 and z is 0 or 1. In a further embodiment, L is orincorporates the formula —O—(CH₂)—. In yet another embodiment, L is orincorporates a phenylene or a 2-carboxy-substituted phenylene.

Any combination of linkers may be used to attach the DYE, Rx or Sc andthe crown ether chelate together, typically a compound of the presentinvention when attached to more than one DYE, Rx or Sc will have one ortwo linkers attached that may be the same or different. The linker mayalso be substituted to after the physical properties of the crown etherchelate compound, such as binding affinity of the chelating moiety andspectral properties of the dye.

Another important feature of the linker is to provide an adequate spacebetween the crown ether chelate moiety and the DYE, Rx or Sc so as toprevent these substituents from providing a steric hindrance to thebinding of the target metal ion for the chelating moiety of the presentcompounds. Therefore, the linker of the present compounds is importantfor (1) attaching DYE, Rx or Sc to the metal chelating moiety, (2)providing an adequate space between DYE, Rx or Sc and the metalchelating moiety so as not to sterically hinder the affinity of thechelating moiety and the zinc ions and (3) for altering the affinity ofthe chelating moiety for the target ions either by the choice of theatoms of the linker or indirectly by addition of substituents to thelinker.

However, it is important to understand that a linker is not an essentialcomponent of the present compounds. Depending on the reporter molecule,a linker may not be necessary wherein the reporter molecule shares atomswith the metal chelating moiety, i.e. a fused DYE. A reporter moleculethat exemplifies this is the dye benzofuran wherein one of the benzenerings of the dye is also one of the benzene rings of the metal chelatingmoiety. In addition, compounds represented for Formula (II) may not besubstituted by a moiety that incorporates a linker.

3. DYE Moiety of the Crown Ether Chelate Compounds

The DYE moiety of the present invention functions as a reporter moleculeto confer a detectable signal, directly or indirectly, to the targetmetal ions. This results in the ability to detect, monitor andquantitate target metal ions in a sample.

The DYE moiety includes without limit a fluorophore, a chromophore, afluorescent protein and an energy transfer pair. When the DYE moiety isa chromophore the crown ether chelate compounds are chromogenicindicators, or more preferably, the DYE moiety is a fluorophore,resulting in a compound that is a fluorogenic indicator for target ions,preferably sodium ions. Therefore, binding a sodium ion with a crownether compound of the resent invention results in a detectable opticalresponse that can be correlated to the presence of sodium ions.

Where the detectable response is a fluorescence response, it istypically a change in fluorescence, such as a change in the intensity,excitation or emission wavelength distribution of fluorescence,fluorescence lifetime, fluorescence polarization, or a combinationthereof. Preferably, the detectable optical response upon binding atarget sodium ion is a change in fluorescence intensity that is greaterthan approximately 10-fold, more preferably greater than 50-fold, andmost preferably more that 100-fold. This large increase in fluorescentsignal over baseline has not been previously observed with other sodiumindicators that comprise a different metal chelating moiety. In anotheraspect, the detectable optical response upon binding the target metalion is a shift in maximal excitation or emission wavelength that isgreater than about 20 nm, more preferably greater than about 30 nm.Sodium and potassium indicators of the type that exhibit significantexcitation and/or emission shifts have not been previously described.

The DYE moiety is any chemical moiety that exhibits an absorptionmaximum beyond 320 nm, that is bound to the crown ether chelate by acovalent linkage L, or that is fused to the crown ether chelate. Apreferred embodiment for detecting sodium ions in live cells is afluorogenic crown ether chelate compound wherein the DYE moiety issubstituted with a lipophilic group. As described above, the covalentlinkage can be a single covalent bond or a combination of stablechemical bonds. The covalent linkage binding the DYE moiety to the crownether chelator is typically a single bond, but optionally incorporates1-20 nonhydrogen atoms selected from the group consisting of C, N, O, P,and S.

A wide variety of chemically reactive fluorescent dyes that may besuitable for incorporation into the compounds of the invention arealready known in the art (RICHARD P. HAUGLAND, MOLECULAR PROBES HANDBOOKOF FLUORESCENT PROBES AND RESEARCH PRODUCTS (Supra); BIOPROBES 32(December 1999); BIOPROBES 33 (February 2000); BIOPROBES 34 (May 2000);and BIOPROBES 35 (November 2000)). The spectral properties of candidatedyes in solution or when conjugated to proteins such as IgG are known orare readily measured using an absorption spectrometer or aspectrofluorometer.

Thus, the DYE moiety of the present invention include, withoutlimitation: a pyrene, an anthracene, a naphthalene, an acridine, astilbene, an indole or benzindole, an oxazole or benzoxazole, a thiazoleor benzothiazole, a 4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), acarbocyanine (including any corresponding compounds in U.S. Ser. No.09/557,275; US Publication Nos. 2002/0077487 and 2002/0064794 and U.S.Pat. Nos. 6,403,807; 6,348,599; 5,486,616: 5,268,486; 5,569,587;5,569,766; 5,627,027 and 6,048,982), a carbostyryl, a porphyin, asalicylate, an anthranilate, an azulene, a perylene, a pyridine, aquinoline, a borapolyazaindacene (including any corresponding compoundsdisclosed in U.S. Pat. Nos. 4,774,339; 5,187,288; 5,248,782; 5,274,113;and 5,433,896), a xanthene (including any corresponding compoundsdisclosed in U.S. Pat. Nos. 6,162,931; 6,130,101; 6,229,055; 6,339,392;5,451,343 and US Publication No. 2002/0059684), an oxazine or abenzoxazine, a carbazine (including any corresponding compoundsdisclosed in U.S. Pat. No. 4,810,636), a phenalenone, a coumarin(including an corresponding compounds disclosed in U.S. Pat. Nos.5,696,157; 5,459,276; 5,501,980 and 5,830,912), a benzofuran (includingan corresponding compounds disclosed in U.S. Pat. Nos. 4,603,209 and4,849,362) and benzphenalenone (including any corresponding compoundsdisclosed in U.S. Pat. No. 4,812,409) and derivatives thereof. As usedherein, oxazines include resorufins (including any correspondingcompounds disclosed in U.S. Pat. No. 5,242,805), aminooxazinones,diaminooxazines, and their benzo-substituted analogs.

Where the DYE moiety is a xanthene, the dye is optionally a fluorescein,a rhodol (including any corresponding compounds disclosed in U.S. Pat.Nos. 5,227,487 and 5,442,045), or a rhodamine (including anycorresponding compounds in U.S. Pat. Nos. 5,798,276 and 5,846,737). Asused herein, fluorescein includes benzo- or dibenzofluoresceins,seminaphthofluoresceins, or naphthofluoresceins. Similarly, as usedherein rhodol includes seminaphthorhodafluors (including anycorresponding compounds disclosed in U.S. Pat. No. 4,945,171).Fluorinated xanthene dyes have been described previously as possessingparticularly useful fluorescence properties (Int. Publ. No. WO 97/39064and U.S. Pat. No. 6,162,931).

Alternatively, the DYE moiety is a xanthene that is bound via an L thatis a single covalent bond at the 9-position of the xanthene. Preferredxanthenes include derivatives of 3H-xanthen-6-ol-3-one bound at the9-position, derivatives of 6-amino-3H-xanthen-3-one bound at the9-position, or derivatives of 6-amino-3H-xanthen-3-imine bound at the9-position.

Preferred DYE moieties of the present invention include xanthene(including rhodol, fluorescein, rhodamine), benzofuran, indole,carbocyanine, quinazolinone, a benzazole, oxazine, coumarin andborapolyazaindacene. The xanthene dyes of this invention comprise bothcompounds substituted and unsubstituted on the carbon atom of thecentral ring of the xanthene by substituents typically found in thexanthene-based dyes such as phenyl and substituted-phenyl moieties. Inaddition, a preferred DYE moiety includes a xanthene-based moiety suchas fluorescein that has a lipophilic group substituted on the oxygenatom. Most preferred dyes are rhodamine, fluorescein,borapolyazaindacene, indole and benzofuran. The choice of the dyeattached to the chelating moiety will determine the crown ether chelatecompound's absorption and fluorescence emission properties as well asits live cell properties, i.e. substituted lipophilic groups

Typically the DYE moiety contains one or more aromatic or heteroaromaticrings, that are optionally substituted one or more times by a variety ofsubstituents, including without limitation, halogen, nitro, sulfo,cyano, alkyl, perfluoroalkyl, alkoxy, alkenyl, alkynyl, cycloalkyl,arylalkyl, acyl, aryl or heteroaryl ring system, benzo, or othersubstituents typically present on chromophores or fluorophores known inthe art.

In one aspect of the invention, the DYE moiety has an absorption maximumbeyond 480 nm. In a particularly useful embodiment, the DYE moietyabsorbs at or near 488 nm to 514 nm (particularly suitable forexcitation by the output of the argon-ion laser excitation source) ornear 546 nm (particularly suitable for excitation by a mercury arclamp). The DYE moiety may be a chromophore, resulting in a compound thatacts as a chromogenic indicator, or more preferably, DYE is additionallya fluorophore, resulting in a compound that is a fluorescent indicator.

Selected sulfonated DYE moieties also exhibit advantageous properties,and include sulfonated pyrenes, coumarins, carbocyanines, and xanthenes(as described in U.S. Pat. Nos. 5,132,432; 5,696,157; 5,268,486;6,130,101). Sulfonated pyrenes and coumarins are typically excited atwavelengths below about 450 nm (U.S. Pat. Nos. 5,132,432 and 5,696,157).

Fluorescent proteins also find use as DYE moieties for the crown etherchelate compounds of the present invention. Examples of fluorescentproteins include green fluorescent protein (GFP) and thephycobiliproteins and the derivatives thereof. The fluorescent proteins,especially phycobiliproteins, are particularly useful for creatingtandem dye-reporter molecules. These tandem dyes comprise a fluorescentprotein and a fluorophore for the purposes of obtaining a larger Stokesshift, wherein the emission spectra are farther shifted from thewavelength of the fluorescent protein's absorption spectra. Thisproperty is particularly advantageous for detecting a low quantity of atarget sodium ion in a sample wherein the emitted fluorescent light ismaximally optimized; in other words, little to none of the emitted lightis reabsorbed by the fluorescent protein. For this to work, thefluorescent protein and fluorophore function as an energy transfer pairwherein the fluorescent protein emits at the wavelength that theacceptor fluorophore absorbs and the fluorophore then emits at awavelength farther from the fluorescent proteins than could have beenobtained with only the fluorescent protein. Alternatively, thefluorophore functions as the energy donor and the fluorescent protein isthe energy acceptor. Particularly useful fluorescent proteins are thephycobiliproteins disclosed in U.S. Pat. Nos. 4,520,110; 4,859,582;5,055,556 and the fluorophore bilin protein combinations disclosed inU.S. Pat. No. 4,542,104. Alternatively, two or more fluorophore dyes canfunction as an energy transfer pair wherein one fluorophore is a donordye and the other is the acceptor dye including any dye compoundsdisclosed in U.S. Pat. Nos. 6,358,684; 5,863,727; 6,372,445; 6,221,606;6,008,379; 5,945,526; 5,863,727; 5,800,996; 6,335,440; 6,008,373;6,184,379; 6,140,494 and 5,656,554.

In one aspect of the invention, the compound of the invention has theformula

where Y is either O or NR⁴; and the DYE moiety is an indole, a coumarin,a stilbene, a xanthene, or a polyazaindacene. In this embodiment,preferably the DYE moiety is a xanthene, a polyazaindacene, or anoxazine.

In one aspect of the invention, the compounds of the invention arefluorescent indicators having the following structure:

wherein the linker is a single covalent bond. These indicators typicallyexhibit a low fluorescence quantum efficiency in the absence of metalions. However, in the presence of increasing metal ion concentration thefluorescence quantum efficiency rises dramatically. For example,selected indicators of this family exhibit a fluorescence signalincrease of over 100-times between zero and a saturating sodiumconcentration. Other selected indicators of the invention exhibit ashift of the wavelength of the absorption (excitation) maximum, emissionmaximum, or both, upon binding the target ion. It appears that havingthe DYE moiety covalently attached or fused to the benzo moiety of thecrown ether chelate compounds provides an additional channel, inaddition to the chelate ring, to conduct electron density changes thatoccur upon metal binding, resulting in larger optical change. This istrue for compounds represented by Formula (I) or (II)

In another aspect of the invention, the compounds of the invention arefluorescent indicators having the following structure:

This class of indicators, in which at least one of the aromatic rings ofthe crown ether portion of the indicator is also incorporated in the DYEmoiety, typically exhibit ratiometric fluorescence excitation changes inresponse to changing metal ion concentration. That is, there is a shiftin the excitation maximum wavelength in the presence of increasing metalion concentrations.

In one embodiment of this aspect, R¹ and R² are C₁-C₆ alkyl that aresubstituted one or more times by cyano, an aryl or heteroaryl ringsystem, or by —(C═O)—O—R¹⁶ or —(C═O)—NR¹⁷R¹⁸, where R¹⁶ is H, a C₁-C₆alkyl, a benzyl, a biologically compatible esterifying group, or abiologically compatible salt; and R¹⁷ and R¹⁸ are independently H, C₁-C₆alkyl, C₁-C₆ carboxyalkyl, an alpha-acyloxymethyl, or a biologicallycompatible salt. The substituents R⁷, R¹⁰, and R¹¹—R¹⁴, areindependently H, chloro, bromo, fluoro, nitro, amino, or cyano; or C₁-C₆alkyl or C₁-C₆ alkoxy that is itself optionally substituted by halogen,amino, hydroxy, —(SO₂)—R¹⁵, —(SO₂)—O—R¹⁵, —(C═O)—R¹⁵, —(C═O)—O—R¹⁶, or—(C═O)—NR¹⁷R¹⁸.

In one embodiment of this aspect, R¹ and R² are C₁-C₆ alkyl that aresubstituted one or more times by cyano, an aryl or heteroaryl ringsystem, or by —(C═O)—O—R¹⁶ or —(C═O)—NR¹⁷R¹⁸, where R¹⁶ is H, a C₁-C₆alkyl, a benzyl, a biologically compatible esterifying group, or abiologically compatible salt; and R¹⁷ and R¹⁸ are independently H, C₁-C₅alkyl, C₁-C₆ carboxyalkyl, an alpha-acyloxymethyl, or a biologicallycompatible salt. The substituents R⁷ R⁸, R¹⁰, and R¹¹—R¹⁴, areindependently H, chloro, bromo, fluoro, nitro, amino, or cyano; or C₁-C₅alkyl or C₁-C₆ alkoxy that is itself optionally substituted by halogen,amino, hydroxy, —(SO₂)—R¹⁵, —(SO₂)—O—R¹⁵, —(C═O)—R¹⁵, —(C═O)—O—R¹⁶, or—(C═O)—NR¹⁷R¹⁸. The DYE moiety is a polyazaindacene, an oxazine, or axanthene, which is optionally substituted one or more times by halogen,nitro, sulfo, cyano, an aryl or heteroaryl ring system, or benzo, oralkyl, perfluoroalkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, arylalkyl,acyl, or carboxylic acids or carboxylic acid esters, the alkyl portionsof which contain fewer than 20 carbons.

An additional selected embodiment of the invention has the formula

where R⁸ and R¹⁶ are defined as above.

An additional selected embodiment of the invention has the formula

or the formula

where R⁸ and R¹⁶ are defined as above, and R²⁶ is H, a C₁-C₆ alkyl, abenzyl, or is an alpha-acyloxyalkyl or a trialkylsilyl or otherbiologically compatible esterifying group, or is a biologicallycompatible salt.

An additional selected embodiment of the invention has the formula

where R¹⁶ is defined as above, and W and W′ are independently F or Cl.

An additional selected embodiment of the invention has the formula

where R⁸ and R¹⁶ are defined as above, and R³⁰—R³⁵ are independentlyhydrogen, halogen, nitro, sulfo, cyano, alkyl, perfluoroalkyl, alkoxy,alkenyl, alkynyl, cycloalkyl, arylalkyl, or acyl, wherein the alkylportions of each contain fewer than 20 carbons; or aryl or heteroarylring system; or adjacent substituents R³¹ and R³², and adjacentsubstituents R³³ and R³⁴, when taken in combination form a fused benzoring that is optionally substituted one or more times by hydrogen,halogen, nitro, sulfo, cyano, alkyl, perfluoroalkyl, alkoxy, alkenyl,alkynyl, cycloalkyl, alkylthio, alkylamido, amino, monoalkylamino ordialkylamino wherein the alkyl portions of each contain fewer than 20carbons.

In another aspect of the invention, the crown ether chelate compoundsare fluorescent indicators represented by the following structure:

wherein Y, P and Q are oxygen and R⁹ or R⁸ is represented by a -L-DYE.Preferably R⁹ is -L-DYE wherein the linker is typically a singlecovalent bond and the DYE moiety is selected from the group consistingof borapolyazaindacene, xanthene and indole. Most preferred are xantheneand indole DYE moieties.

A particularly preferred embodiment of this aspect is represent by thefollowing formula:

wherein R¹⁶ is selected from the group consisting of H, C₁-C₆ alkyl,benzyl, a biologically compatible esterifying group, and a biologicallycompatible salt. Preferably R¹⁶ is methyl, a biologically compatibleesterifying group or a biologically compatible salt.

R¹⁹ and R²⁰ are selected from the group consisting of H, halogen, azido,nitro, nitroso, amino, cyano, -L-R_(x), -L-S_(c), -L-DYE, C₁-C₆ alkyland C₁-C₆ alkoxy, each of which is itself optionally substituted byhalogen, amino, hydroxy, —(SO₂)—R¹⁵, —(SO₂)—O—R¹⁵, —(C═O)—R¹⁵,—(C═O)—O—R¹⁶ and —(C═O)—NR¹⁷R¹⁸. Alternatively, R¹⁹ and R²⁰ taken incombination from a fused six-membered benzo moiety that is optionallysubstituted by halogen, azido, nitro, nitroso, amino, cyano, -L-R_(x),-L-S_(c), -L-DYE, C₁-C₆ alkyl or C₁-C₆ alkoxy, each of which is itselfoptionally substituted by halogen, amino, hydroxy, —(SO₂)—R¹⁵,—(SO₂)—O—R¹⁵, —(C═O)—R¹⁵, —(C═O)—O—R¹⁶, or —(C═O)—NR¹⁷R¹⁸. Typically,R¹⁹ and R²⁰ are hydrogen or taken together form a fused benzo moiety.

R⁷, R⁸, and R¹⁰ are independently selected from the group consisting ofH, halogen, azido, nitro, nitroso, amino, cyano, -L-R_(x), -L-S_(c),-L-DYE, C₁-C₆ alkyl and C₁-C₈ alkoxy, each of which is optionallysubstituted by halogen, amino, hydroxy, —(SO₂)—R¹⁵, —(SO₂)—O—R¹⁵,—(C═O)—R¹⁵, —(C═O)—O—R¹⁶, or —(C═O)—NR¹⁷R¹⁸. Alternatively, R⁷ taken incombination with R⁸ form a fused six-membered benzo moiety, which isoptionally substituted by halogen, azido, nitro, nitroso, amino, cyano,-L-R_(x), -L-S_(c), -L-DYE, C₁-C₆ alkyl or C₁-C₆ alkoxy, each of whichis optionally substituted by halogen, amino, hydroxy, —(C═O)—R¹⁵,—(C═O)—O—R¹⁶, or —(C═O)—NR¹⁷R¹⁸. Typically, R⁷, R⁸ and R¹⁰ are hydrogen.

R²¹ is selected from the group consisting of H, C₁-C₁₈ alkyl, C₇-C₁₈arylalkyl and lipophilic group each alkyl is optionally substituted by—(C═O)—R¹⁵, —(C═O)—O—R¹⁶, or C₁-C₈ alkoxy.

A particularly preferred embodiment is compound 86 and 115 and thecorresponding cell-permanent versions, compounds 87 and 116 (See,Examples 79, 80, 105 and 106).

In another aspect of the invention, the crown ether chelate compoundsare represented by the formula:

wherein R⁹ or R⁸ are -L-DYE, preferably R⁹ is -L-DYE wherein L istypically a single covalent bond and the DYE moiety isborapolyazaindacene, xanthene or indole. Most preferred are xanthene andindole DYE moieties. Y, P and Q are oxygen. E² and E³ are —(CH₂)₂— andE¹ is —(CH₂)O(CH₂)—.

A preferred embodiment is represented by compound 109 and thecell-permanent version; compound 110 (See, Examples 99 and 100).

In another aspect of the invention, the crown ether chelate compoundsare represented by the formula:

wherein R⁹ or R⁸ are -L-DYE, preferably R⁹ is -L-DYE, wherein L istypically a single covalent bond and the DYE moiety isborapolyazaindacene, xanthene or indole. Most preferred is a xantheneDYE moiety. P and Q are oxygen and Y is absent. E¹ and E³ are —(CH₂)₂—and E² is absent.

Typically R⁷, R⁸ or R⁹, R¹⁰, R¹⁹ and R²⁰ are hydrogen and R¹ is a methylor ethyl group that is substituted by —(C═O)—R¹⁵ or —(C═O)—O—R¹⁵ whereinR¹⁵ and R¹⁶ are methyl or ethyl.

A preferred embodiment is represented by compound 104 (See, Example 94).

The family of crown ether chelate compounds represented by Formula (II)are particularly useful for binding physiological relevant levels ofmetal ions in vivo wherein the DYE moiety is substituted by an AM oracetate ester. In addition, this family of compounds unexeptedly crosslive cell membranes easier that compounds represented by Formula (I) andare thus preferred for binding of target metal ions in live cells.Furthermore, compounds represented by Formula (II), wherein the secondnitrogen atom has been replaced by an oxygen atom, unexeptedly resultedin a higher affinity binding of target metal ions such as sodium ions.This, in combination with their ability to effectively load into livecells, provides for unexpected advantages over compounds represented byFormula (I) for in vivo binding of target ions.

The family of crown ether chelate compounds represented by Formula (II)are also very useful for the binding and detection of target metal ionsin vitro (See, Table 2). These compounds demonstrate a significantchange in fluorescent signal after binding the target metal ions.

Selected embodiments of the invention are given in Table 1, showing avariety of distinct DYE moieties and crown ether substituents useful inthe invention.

TABLE 1 Selected embodiments of the invention for Formula (I) compounds.Dissociation Constant Compound (target ion)

K_(d) (Na⁺) = ~52 mM K_(d) (K⁺) = ~330 mM Compound 51

K_(d) (Na⁺) = ~30 mM K_(d) (K⁺) = ~115 mM Compound 34

K_(d) (Na⁺) = ~220 mM Compound 63

K_(d) (Na⁺) = ~52 mM K_(d) (K⁺) = ~250 mM Compound 57

K_(d) (Na⁺) = ~60 mM K_(d) (K⁺) = ~205 mM Compound 22

K_(d) (Na⁺) = ~42 mM Compound 32

K_(d) (Na⁺) = ~28 mM K_(d) (K⁺) = ~130 mM Compound 79

K_(d) (Na⁺) = ~95 mM K_(d) (K⁺) = ~300 mM Compound 27

K_(d) (Na⁺) = ~92 mM K_(d) (K⁺) = ~705 mM Compound 38

K_(d) (Na⁺) = ~70 mM K_(d) (Zn²⁺) = ~100 mM Compound 60

K_(d) (Na⁺) = ~85 mM K_(d) (K⁺) = ~255 mM Compound 78

K_(d) (Na⁺) = ~14 mM Compound 42

K_(d) (Na⁺) = ~103 mM K_(d) (K⁺) = ~205 mM Compound 30

K_(d) (Zn²⁺) = ~300 nM K_(d) (Ca²⁺) = ~4 μM K_(d) (Na⁺) = ~38 mM K_(d)(K⁺) = ~270 mM Compound 28

K_(d) (Zn²⁺) = ~8 μM K_(d) (Ca²⁺) = ~7 μM Compound 31

K_(d) (Na⁺) = ~36 mM K_(d) (K⁺) = ~215 mM K_(d) (Zn²⁺) = ~300 nM K_(d)(Ca²⁺) = ~5 μM Compound 23

K_(d) (Na⁺) = ~2.0 M K_(d) (Zn²⁺) = ~600 μM Compound 79

K_(d) (Na⁺) = ~500 mM K_(d) (Zn²⁺) = ~650 μM Compound 65

K_(d) (Ca²⁺) = ~400 nM K_(d) (Na⁺) = ~160 mM Compound 54

Compound 80

Compound 81

Selected preferred embodiments for in solution binding of target ionswith compounds represented by Formula (II):

TABLE 2 Dissociation constant (K_(d)) and Response to complexationCompound # Dye moiety R¹ = (R = F/F₀) for Target metal ions 104 xanthene—CH₂COOCH₃ K_(d) (Li⁺) = 438 mM; R (Li⁺) = 2.8 K_(d) (Na⁺) = 226 mM; R(Na⁺) = 19.7 K_(d) (K⁺) = 978 mM; R (K⁺) = 9.7 K_(d) (Rb⁺) = 376 mM; R(Rb⁺) = 2.9 86 xanthene —CH₂COOCH₃ K_(d) (Li⁺) = 142 mM; R (Li⁺) = 6.8K_(d) (Na⁺) = 82 mM; R (Na⁺) = 28.5 K_(d) (K⁺) = 291 mM; R (K⁺) = 6.9K_(d) (Rb⁺) = 319 mM; R (Rb⁺) = 2.7 110 xanthene —CH₂COOCH₃ K_(d) (Li⁺)= no sensitivity K_(d) (Na⁺) = 15 mM; R (Na⁺) = 1.5 K_(d) (K⁺) = 11 mM;R (K⁺) = 2.2 K_(d) (Rb⁺) = 25 mM; R (Rb⁺) = 1.5 93 indole —CH₂COOCH₃K_(d) (Na⁺) = 89 mM; R (Na⁺) = 25 K_(d) (K⁺) = no sensitivity 89xanthene —CH₂COOCH₃ K_(d) (Na⁺) = 163 mM; R (Na⁺) = 41 K_(d) (K⁺) = 254mM; R (K⁺) = 10.6 90 borapolyazaindacene —CH₂COOCH₃ K_(d) (Na⁺) = 100mM; R (Na⁺) = 7.3 K_(d) (K⁺) = 170 mM; R (K⁺) = 2.9 127 xanthene—CH₂COOH K_(d) (Na⁺) = 20 mM; R (Na⁺) = 3.6 115 xanthene —(CH₂)₂COCH₃K_(d) (Na⁺) = 78.8 mM; R (Na⁺) = 5.9 K_(d) (K⁺) = 269 mM; R (K⁺) = 2.8118 xanthene —(CH₂)₂COCH₃ K_(d) (Na⁺) = 150 mM; R (Na⁺) = 12 120 indole—(CH₂)₂COCH₃ K_(d) (Na⁺) = 89 mM; R (Na⁺) = 2 126 indole —(CH₂)₂CN(CH₃)₂K_(d) (Na⁺) = 379 mM; R (Na⁺) = −1.2 99 xanthene —CH₂COOCH₃ K_(d) (Li⁺)= 65 mM; R (Li⁺) = 3.1 K_(d) (Na⁺) = 59 mM; R (Na⁺) = 3.9 K_(d) (K⁺) =144 mM; R (K⁺) = 2.7 K_(d) (Rb⁺) = 157 mM; R (Rb⁺) = 1.9

4. Reactive Functional Groups and Conjugated Substances of the CrownEther Chelate Compounds.

As described above, the compounds of the invention may be substitutedone or more times by a -L-R_(x) moiety or -L-S_(c) moiety. L is acovalent linkage that is a single covalent bond or a series of stablebonds comprising 1-20 nonhydrogen atoms selected from the groupconsisting of C, O, N, P and S. R_(x) is a reactive group that functionsas the site of attachment for another moiety wherein the reactive groupchemically reacts with an appropriate reactive or functional group onanother substance or moiety. These reactive groups are synthesizedduring the formation of the present compounds providing present crownether chelate compounds that can be covalently attached to anothersubstance, conjugated substance, facilitated by the reactive group. Inthis way, compounds incorporating a reactive group (R_(x)) can becovalently attached to a wide variety of biomolecules ornon-biomolecules that contain or are modified to contain functionalgroups with suitable reactivity, resulting in chemical attachment of theconjugated substance (S_(c)), represented by -L-S_(c). The reactivegroup and functional group are typically an electrophiles and anucleophile that can generate a covalent linkage. Alternatively, thereactive group is a photoactivatable group, and becomes chemicallyreactive only after illumination with light of an appropriatewavelength. Typically, the conjugation reaction between the reactivegroup and the substance to be conjugated results in one or more atoms ofthe reactive group R_(x) to be incorporated into a new linkage attachingthe compound of the invention to the conjugated substance S_(c).Selected examples of functional groups and linkages are shown in Table3, where the reaction of an electrophilic group and a nucleophilic groupyields a covalent linkage.

TABLE 3 Examples of some routes to useful covalent linkagesElectrophilic Group Nucleophilic Group Resulting Covalent Linkageactivated esters* amines/anilines carboxamides acrylamides thiolsthioethers acyl azides** amines/anilines carboxamides acyl halidesamines/anilines carboxamides acyl halides alcohols/phenols esters acylnitriles alcohols/phenols esters acyl nitriles amines/anilinescarboxamides aldehydes amines/anilines imines aldehydes or ketoneshydrazines hydrazones aldehydes or ketones hydroxylamines oximes alkylhalides amines/anilines alkyl amines alkyl halides carboxylic acidsesters alkyl halides thiols thioethers alkyl halides alcohols/phenolsethers alkyl sulfonates thiols thioethers alkyl sulfonates carboxylicacids esters alkyl sulfonates alcohols/phenols ethers anhydridesalcohols/phenols esters anhydrides amines/anilines carboxamides arylhalides thiols thiophenols aryl halides amines aryl amines aziridinesthiols thioethers boronates glycols boronate esters carbodiimidescarboxylic acids N-acylureas or anhydrides diazoalkanes carboxylic acidsesters epoxides thiols thioethers haloacetamides thiols thioethershaloplatinate amino platinum complex haloplatinate heterocycle platinumcomplex haloplatinate thiol platinum complex halotriazinesamines/anilines aminotriazines halotriazines alcohols/phenols triazinylethers halotriazines thiols triazinyl thioethers imido estersamines/anilines amidines isocyanates amines/anilines ureas isocyanatesalcohols/phenols urethanes isothiocyanates amines/anilines thioureasmaleimides thiols thioethers phosphoramidites alcohols phosphite esterssilyl halides alcohols silyl ethers sulfonate esters amines/anilinesalkyl amines sulfonate esters thiols thioethers sulfonate esterscarboxylic acids esters sulfonate esters alcohols ethers sulfonylhalides amines/anilines sulfonamides sulfonyl halides phenols/alcoholssulfonate esters *Activated esters, as understood in the art, generallyhave the formula —COΩ, where Ω is a good leaving group (e.g.,succinimidyloxy (—OC₄H₄O₂) sulfosuccinimidyloxy (—OC₄H₃O₂—SO₃H),-1-oxybenzotriazolyl (—OC₆H₄N₃); or an aryloxy group or aryloxysubstituted one or more times by electron withdrawing substituents suchas nitro, fluoro, chloro, cyano, or trifluoromethyl, or combinationsthereof, used to form activated aryl esters; or a carboxylic acidactivated by a carbodiimide to form an anhydride or mixed anhydride—OCOR^(a) or —OCNR^(a)NHR^(b), where R^(a) and R^(b), which may be thesame or different, are C₁-C₆ alkyl, C₁-C₆ perfluoroalkyl, or C₁-C₆alkoxy; or cyclohexyl, 3-dimethylaminopropyl, or N-morpholinoethyl).**Acyl azides can also rearrange to isocyanates

Choice of the reactive group used to attach the compound of theinvention to the substance to be conjugated typically depends on thereactive or functional group on the substance to be conjugated and thetype or length of covalent linkage desired. The types of functionalgroups typically present on the organic or inorganic substances(biomolecule or non-biomolecule) include, but are not limited to,amines, amides, thiols, alcohols, phenols, aldehydes, ketones,phosphates, imidazoles, hydrazines, hydroxylamines, disubstitutedamines, halides, epoxides, silyl halides, carboxylate esters, sulfonateesters, purines, pyridines, carboxylic acids, olefinic bonds, or acombination of these groups. A single type of reactive site may beavailable on the substance (typical for polysaccharides or silica), or avariety of sites may occur (e.g., amines, thiols, alcohols, phenols), asis typical for proteins. A conjugated substance may be conjugated tomore than one crown ether chelate compound, which may be the same ordifferent, or to a substance that is additionally modified by a hapten,such as biotin. Although some selectivity can be obtained by carefulcontrol of the reaction conditions, selectivity of labeling is bestobtained by selection of an appropriate reactive functional group.

Typically, R_(x) will react with an amine, an alcohol, an aldehyde, aketone, or with silica. Preferably R_(x) reacts with an amine or a thiolfunctional group, or with silica. In one embodiment, R_(x) is anacrylamide, an activated ester of a carboxylic acid, an acyl azide, anacyl nitrile, an aldehyde, an alkyl halide, a silyl halide, ananhydride, an aniline, an aryl halide, an azide, an aziridine, aboronate, a diazoalkane, a haloacetamide, a halotriazine, a hydrazine(including hydrazides), an imido ester, an isocyanate, anisothiocyanate, a maleimide, a phosphoramidite, a reactive platinumcomplex, a sulfonyl halide, or a thiol group. By “reactive platinumcomplex” is particularly meant chemically reactive platinum complexessuch as described in U.S. Pat. No. 5,714,327.

Where R_(x) is an activated ester of a carboxylic acid, the resultingcompound is particularly useful for preparing conjugates of proteins,nucleotides, oligonucleotides, or haptens. Where R_(x) is a maleimide orhaloacetamide the resulting compound is particularly useful forconjugation to thiol-containing substances. Where R_(x) is a hydrazide,the resulting compound is particularly useful for conjugation toperiodate-oxidized carbohydrates and glycoproteins, and in addition isan aldehyde-fixable polar tracer for cell microinjection. Where R_(x) isa silyl halide, the resulting compound is particularly useful forconjugation to silica surfaces, particularly where the silica surface isincorporated into a fiber optic probe subsequently used for remote iondetection or quantitation.

Preferably, R_(x) is a succinimidyl ester of a carboxylic acid, ahaloacetamide, a hydrazine, an isothiocyanate, a maleimide group, analiphatic amine, a silyl halide, or a psoralen. More preferably, R_(x)is a succinimidyl ester of a carboxylic acid, a maleimide, aniodoacetamide, or a silyl halide. In a particular embodiment R_(x) is asilyl halide or an isothiocyanate.

The compounds of the invention that possess a reactive functional groupare useful for conjugation to any substance that possesses a suitablefunctional group for covalent attachment of the chelate. Examples ofparticularly useful conjugates include, among others, conjugates ofantigens, steroids, vitamins, drugs, haptens, metabolites, toxins,environmental pollutants, amino acids, peptides, proteins, nucleicacids, nucleic acid polymers, carbohydrates, lipids, and non-biologicalpolymers. Alternatively, these are conjugates of cells, cellularsystems, cellular fragments, or subcellular particles. Examples include,among others, virus particles, bacterial particles, virus components,biological cells (such as animal cells, plant cells, bacteria, yeast, orprotists), or cellular components.

Preferably the conjugated substance is a protein, polysaccharide, lipid,lipid assembly, non-biological polymer, or polymeric microparticle.Another class of preferred conjugated substances includes particles orfibers composed of silica or other glasses, useful for preparing opticaldevices for remote sensing.

Preferred nucleic acid polymer conjugates are labeled, single- ormulti-stranded, natural or synthetic DNA or RNA, DNA or RNAoligonucleotides, or DNA/RNA hybrids, or incorporate an unusual linkersuch as morpholine derivatized phosphates (AntiVirals, Inc., CorvallisOreg. ), or peptide nucleic acids such as N(2-aminoethyl)glycine units.

In a preferred embodiment, the conjugated substance (S_(c)) is acarbohydrate that is typically a polysaccharide, such as a dextran,FICOLL, heparin, glycogen, amylopectin, mannan, inulin, starch, agaroseand cellulose. Alternatively, the carbohydrate is a polysaccharide thatis a lipopolysaccharide. Preferred polysaccharide conjugates aredextran, FICOLL, or lipopolysaccharide conjugates.

In another embodiment, the conjugated substance (S_(c)), is a lipidmoiety (typically having 6-60 carbons), including glycolipids,phospholipids, sphingolipids, and steroids. Alternatively, theconjugated substance is a lipid assembly, such as a liposome. The lipidmoiety may be used to retain the conjugated substances in cells, asdescribed in U.S. Pat. No. 5,208,148.

Other conjugates of non-biological materials include conjugates oforganic or inorganic polymers, polymeric films, polymeric wafers,polymeric membranes, polymeric particles, or polymeric microparticle,including magnetic and non-magnetic microspheres, conducting andnon-conducting metals and non-metals, and glass and plastic surfaces andparticles. Conjugates are optionally prepared by copolymerization of acompound of the invention that contains an appropriate functionalitywhile preparing the polymer, or by chemical modification of a polymerthat contains functional groups with suitable chemical reactivity. Othertypes of reactions that are useful for preparing conjugates of polymersinclude catalyzed polymerizations or copolymerizations of alkenes andreactions of dienes with dienophiles, transesterifications ortransaminations. In another embodiment the conjugated substance is aglass or silica, which may be formed into an optical fiber or otherstructure.

Conjugates typically result from mixing appropriate reactive dyes andthe substance to be conjugated in a suitable solvent in which both aresoluble. Labeling of insoluble polymers or silica can be performed in asuspension of the insoluble polymer in a suitable solvent. For thosereactive groups that are photoactivated, conjugation requiresillumination of the reaction mixture to activate the reactive group.

Synthesis

There are typically three components to the methodology used to preparethe compounds of the invention. The first involves the formation of thecrown ether chelate itself, the second involves the appropriatederivatization of the secondary amine nitrogen atom(s) of the crownether chelate, and the third, when needed, involves modification of thecrown ether chelate by forming a reactive functional group, covalentlyattaching a conjugate, or covalently attaching a DYE moiety to form anindicator. It should be understood that the DYE moiety is typically notattached to the crown ether chelate compound but that the conjugatedsubstance is attached in this way. Although these synthetic componentsare typically performed in the order given, they may be carried out inany other suitable sequence. For example, a portion of the chelate maybe derivatized with a fluorescent dye prior to formation of the completechelate ring.

Where the P and Q moieties are both oxygen, and E¹ is ethylene, thecrown ether chelate is typically prepared by acylation of abis-(2-aminophenoxy)ethane with a bis-(acid chloride), such asdiglycolyl chloride, followed by reduction of the resulting bis-amide tothe corresponding bis-secondary amine. Selection of the appropriatebis-acid chloride results in the particular desired crown ether (as inExamples 64-69 and 70).

The secondary amine nitrogen atom(s) present in the crown ether aretypically derivatized with an alkylating agent. As the metal bindingability of the resulting crown ether is significantly influenced by thenature of the amine substituents, careful selection of the alkylatingagent may be necessary to prepare a reporter for a particular targetion. Where the crown nitrogens are alkylated by methyl bromoacetate, theresulting bis-aza-crown ether is typically selective for sodium ions. Ifthe alkylating agent is 2-picolyl chloride, the resulting crown ether istypically selective for zinc ions. As discussed above, the presence ofesters vs. carboxylic acids on the amine nitrogen substituents mayinfluence the relative binding affinity of selected target ions.Selection of an alkylating agent that incorporates a precursor to areactive functional group is useful for producing chemically reactivecompounds of the invention, as well as acting as a useful intermediatefor preparing conjugates, as described above. Additionally, analkylating agent that incorporates a reporter DYE results in a crownether compound that functions as an indicator for selected target ions.

More typically, the crown ether chelate is derivatized at the benzo ringof the crown ether. As described above, typically a suitable crown etheris prepared, and then bound to a DYE moiety. In one aspect of theinvention, an ortho-hydroxy aromatic aldehyde is treated with achloromethyl heterocycle to yield a fused reporter (as described inExample 46). In another aspect of the invention, derivatization with aDYE moiety is carried out by modifying a crown ether that possesses analdehyde or ketone functional group.

In one aspect of the invention, the crown ether is substituted by analdehyde and the fluorophore precursors and the crown ether are combinedunder anaerobic or non-oxidative conditions (e.g., under nitrogen), andsubsequently oxidized using a mild oxidant (e.g., a quinone oxidant,preferably DDQ or chloranil). Where xanthene fluorophore precursors arecondensed under anaerobic conditions, the resulting fluorophore is thenon-fluorescent dihydro species, which may be utilized without prioroxidation as a sensor for oxidative subenvironments, e.g., in cells.

In yet another aspect of the invention, the crown ether is substitutedby a carboxylic acid or by an aldehyde that is converted to thecarboxylic acid in the course of synthesis of the crown ether. Thechelating moiety is then condensed with the fluorophore precursors toyield the resulting indicator directly.

Synthesis of conventional xanthene dyes such as fluoresceins, rhodaminesand rhodols typically involve the condensation of two equivalents ofresorcinol (for fluoresceins), aminophenol (for rhodamines) or a mixtureof a resorcinol and an aminophenol (for rhodols) with acarbonyl-containing moiety such as a phthalic acid derivative orbenzaldehyde. In the synthesis of the xanthene indicators of theinvention, the desired resorcinol or aminophenol is condensed with thesubstituted crown ether, yielding either the reduced xanthene (where thecrown ether contains an aldehyde) or the oxidized xanthene (where thecrown ether contains a carboxylic acid or acyl halide) bound directly tothe chelating moiety.

An oxidation step is typically required after condensation of aformyl-substituted crown ether with the fluorophore precursors.Optionally, the dihydro condensation product is isolated andsubsequently oxidized with air or by standard chemical oxidants, such aschloranil. For some fluorophores, the oxidation reaction is enhanced byacidic reaction conditions. These mild oxidation reaction conditionstolerate a wide variety of substituents on the fluorophore and/or crownether of the resulting indicators.

Unsymmetrical xanthene dyes are typically constructed by statisticalmethods, using a 1:1 mixture of the desired resorcinols or aminophenolsin the condensation reaction, and purifying the desired product from thestatistical mix of products using methods known in the art.

The synthesis of polyazaindacene dyes, particularly dipyrometheneborondifluoride dyes, has been well documented (U.S. Pat. Nos. 4,774,339;5,187,288; 5,248,782; 5,274,113; 5,338,854 and 5,433,896). The proceduretypically consists of an acid-catalyzed condensation of a benzaldehydewith a pyrrole that has a hydrogen at the 2-position, followed by insitu oxidation of the condensed intermediate by air, oxygen or achemical oxidant such as 2,3-dichloro-5,6-dicyano-1,4-benzoquinone(DDQ). The condensation of two appropriately substituted pyrroles, eachhaving a hydrogen at the 2-position, with a formyl-substituted crownether, followed by in situ oxidation of the condensed intermediate andtreatment with a boron trifluoride etherate yields thedipyrrometheneboron difluoride indicators of the invention.Alternatively, the indicators are formed via the direct condensation ofa carboxyl- or chlorocarbonyl-substituted crown ether with twoequivalents of appropriately substituted pyrroles, which may be the sameof different, provided each has a hydrogen at the 2-position. The latterprocedure does not require oxidation.

Post-condensation modifications of both the crown ether and thefluorophore moiety are typically strictly analogous to known methods ofindicator modification; for example, the reduction of nitro substituentsto amino groups, the conversion of carboxy substituents to cyano groups,and the preparation of esters of carboxylic acids, includingacetoxymethyl esters. Additionally, salts and counterions of theindicators of the invention are readily converted to other salts bytreatment with ion-exchange resins, selective precipitation, andbasification, as is well-known in the art.

Post-condensation modifications of xanthylium dyes are well known. Forinstance, the xanthenone portion of the dye can be halogenated bytreatment with the appropriate halogenating agent, such as liquidbromine. Xanthenes containing unsaturated fused rings can behydrogenated to the saturated derivatives.

The reduced and oxidized versions of the xanthene indicators are freelyinterconverible by well-known oxidation or reduction reagents, includingborohydrides, aluminum hydrides, hydrogen/catalyst, and dithionites.Care must be exercised to select an oxidation or reducing agent that iscompatible with the crown ether chelator. A variety of oxidizing agentsmediate the oxidation of dihydroxanthenes, including molecular oxygen inthe presence or absence of a catalyst, nitric oxide, peroxynitrite,dichromate, triphenylcarbenium and chloranil. The dihydroxanthenes arealso oxidized electrochemically, or by enzyme action, includinghorseradish peroxidase in combination with peroxides or by nitric oxide.

Rather than condensing the DYE moiety precursors directly with asubstituted crown ether, the preformed DYE moiety may be covalentlybound to the crown ether via a conventional cross-linking reaction. Awide variety of chemically reactive or potentially chemically reactiveand fluorescent fluorescein, rhodamine, rhodol, benzoxanthenes,dibenzoxanthene and other xanthene oxygen heterocycles that absorbmaximally beyond about 490 nm are commercially available as described byHaugland, HANDBOOK OF FLUORESCENT PROBES AND RESEARCH PRODUCTS (Supra),as described above, or in other literature references. The nature of thebond that links the DYE moiety to the crown ether chelate appears tohave an effect on the optical response of the DYE moiety to ion binding,sometimes a significant effect. Acceptability of the linking chemistrycan be determined by titration of the resultant indicator with the ionof interest over the target range of response (as described in Example71).

B. Method of Use

The crown ether compounds of the invention are useful for anyapplication where it is desirable to complex a target metal ion.Selected crown ether compounds of the invention may be useful asionophores, that is, they facilitate the transport of selected targetions across cell membranes. Where the crown ether compound is bound to aconjugated substance that is a polymeric matrix, such as amicroparticle, or agarose, the compounds are useful for depleting asample solution of a selected target ion, particularly where thepolymeric matrix is used to pack a chromatography column. Other crownether compounds (those bound to a DYE moiety) are useful as calorimetricor fluorescent indicators for a selected target ion.

In order for a particular indicator of the present invention to beuseful for detection purposes, it must exhibit a detectable change inspectral properties upon complexation of the desired metal ion (targetion) in the chelating moiety. Preferably the change in spectralproperties is a change in fluorescence properties. More preferably, theinstant indicators display an intensity increase or decrease in emissionenergy upon the complexation of the desired target ion.

However, it should be appreciated that at least the compoundsrepresented by Formula (II) do not require a DYE moiety. These compoundsare useful for binding target ions resulting in a complex of the targetion and the present crown ether chelate compounds. Therefore, anadditional aspect of the invention includes the compound of theinvention further comprising a metal ion that is associated and/orcomplexed within the crown ether chelate portion of the compound. Themetal ion is optionally a monocation (such as Li⁺, Na⁺, K⁺, Rb⁺, orCs⁺), a dication (such as Ca²⁺, Zn²⁺, or Mg²⁺), or a polycation (such asTb³⁺ or Eu³⁺). Preferably the crown ether chelate metal in complexcomprises physiological relevant cations such as sodium, potassium,calcium and zinc.

Accordingly, a method for binding target metal ions in a samplecomprises the following steps:

-   -   a) contacting said sample with a crown ether chelate compound of        the present invention; and,    -   b) incubating said sample and said metal chelating compound for        sufficient time to allow said compound to chelate said target        metal ion whereby said metal ion is bound.

When the present compounds are used as indicators a DYE moiety iscovalently attached to the crown ether chelate. The sample isilluminated with an appropriate wavelength whereby the target ion isdetected. In such an assay the target ion can also be quantitated andmonitored.

The specific indicator used in an assay or experiment is selected basedon the desired affinity for the target ion as determined by the expectedconcentration range in the sample, the desired spectral properties, andthe desired selectivity. Initially, the suitability of a material as anindicator of ion concentration is commonly tested by mixing a constantamount of the indicating reagent with a measured amount of the targetion under the expected experimental conditions.

Preferred indicators display a high selectivity, that is, they show asufficient rejection of non-target ions. The interference of anon-target ion is tested by a comparable titration of the indicator withthat ion. Although preferred target ions for most indicators of thepresent invention are Na⁺ and K⁺, any ion that yields a detectablechange in absorption wavelengths, emission wavelengths, fluorescencelifetimes or other measurable optical property over the concentrationrange of interest is potentially measured using one of the indicators ofthis invention. Modifications to the electronic structure of the crownether or indicator to produce an indicator having the appropriatecombination of binding affinity, ion selectivity and spectral responsefor a wide variety of metal ions.

In one embodiment of the invention, the target ions for the indicatorsof the present invention are selected from Li⁺, Na³⁰ , K⁺, Cs⁺, Ca²⁺,Zn²⁺, Mg²⁺, Rb⁺, Tb³⁺ or Eu³⁺. In another embodiment of the invention,the target ions are selected from Li⁺, Na⁺, K⁺, Ca²⁺, Zn²⁺, and Mg²⁺.Additional target ions for selected embodiments of the presentindicators also include Mn²⁺, Fe²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Cu+, Zn²⁺,Al³⁺, Cd²⁺, Ag⁺, Au⁺, Tl⁺, Pd²⁺, Hg⁺, Sn²⁺, Pb²⁺, Sr²⁺, Ba²⁺, Mo³⁺,Ga³⁺, In³⁺, La³⁺, Eu³⁺, Tb³⁺, Dy³⁺, Ru³⁺, Sc³⁺, As³⁺, Sb³⁺, Cr³⁺, Bi³⁺,Ce³⁺, Ce⁴⁺, Pd²⁺, Pt²⁺ and Pt⁴⁺ ions. In yet another embodiment of theinvention, the target ions of the instant indicators are Fe²⁺, Fe³⁺,Co²⁺, Ni²⁺, Cu²⁺, Cu⁺, Zn²⁺, Al³⁺, Cd²⁺, Hg²⁺, Pd²⁺, Ba²⁺, La³⁺, Tb³⁺and Cr³⁺ ions. In yet another embodiment, the target ions are selectedfrom the group consisting of Fe³⁺, Ni²⁺, Cu²⁺, Cu⁺, Hg²⁺, or Pb²⁺.

The indicator is generally prepared for use as a detection reagent bydissolving the indicator in solution at a concentration that is optimalfor detection of the indicator at the expected concentration of thetarget ion. Modifications that are designed to enhance permeability ofthe indicator through the membranes of living cells, such asacetoxymethyl esters and acetates, may require the indicator to bepredissolved in an organic solvent such as dimethylsulfoxide (DMSO)before addition to a cell suspension, where the indicators then readilyenter the cells. Intracellular enzymes cleave the esters to the morepolar acids and phenols that are then well retained inside the cells.For applications where permeability of cell-membranes is required, theindicators of the invention are typically substituted by only onefluorophore.

Therefore, a method for binding and detecting target ions in a live cellcomprises the following steps:

-   -   a) contacting a sample of live cells with a crown ether chelate        compound of the present invention wherein said compound        comprises a DYE moiety and at least one lipophilic group;    -   b) incubating said sample and said crown ether chelate compound        for sufficient time to allow said compound to chelate said        target metal ion; and,    -   c) illuminate said sample with an appropriate wavelength whereby        said target ion is detected in a live cell.

Typically, the lipophilic group is an AM or acetate ester that isdirectly attached to the DYE moiety of the crown ether chelate compound.

A specific indicator of the present invention is useful for thedetection and/or quantification of a desired target ion, when thebinding of the target ion in the metal ion-binding moiety of theindicator results in a detectable change in spectral properties.Preferably, the change in spectral properties is a detectablefluorescence response.

A preferred indicator for a specific target ion is an indicator thatshows at least a two-fold change in net fluorescence emission intensity(either higher or lower), or a 1 nanosecond difference in fluorescencelifetime (either shorter or longer), preferably a five-fold or greaterchange in net fluorescence emission intensity or a 100% change influorescence lifetime in response to the target ion. Alternatively, anindicator that exhibits a shift in excitation or emission wavelength ofat least 10 nm (either to shorter or longer wavelength) is alsopreferred, more preferably exhibiting a shift of 25 nm or greater.

The optical response of the indicating reagent is determined by changesin absorbance or fluorescence, preferably fluorescence. If absorbancemeasurements are used to determine ion concentrations, then it isusually optimal to adjust the optical density of the indicator in thesample over the range of analyte concentration to a value ofapproximately 0.02 to 2.5 (most preferably 0.1 to 1). For fluorescencemeasurements, the concentration of the indicator will depend mostly onthe sensitivity of the equipment used for its detection.

If the optical response of the indicator will be determined usingfluorescence measurements, samples are typically stained with indicatorconcentrations of 10⁻⁹ M to 10⁻² M. The most useful range of analyteconcentration is about one log unit above and below the dissociationconstant of the ion-indicator complex. This dissociation constant isdetermined by titration of the indicator with a known concentration ofthe target ion, usually over the range of virtually zero concentrationto approximately 100 millimolar of the target ion, depending on whichion is to be measured and which indicator is being used. Thedissociation constant may be affected by the presence of other ions,particularly ions that have similar ionic radii and charge. It may alsobe affected by other conditions such as ionic strength, pH, temperature,viscosity, presence of organic solvents and incorporation of the sensorin a membrane or polymeric matrix, or conjugation or binding of thesensor to a protein or other biological molecule. Any or all of theseeffects need to be taken into account when calibrating an indicator.

The indicator is combined with a sample in a way that will facilitatedetection of the target ion concentration in the sample. The sample isgenerally a representative cell population, fluid or liquid suspensionthat is known or suspected to contain the target ion. Representativesamples include intracellular fluids such as in blood cells, culturedcells, muscle tissue, neurons and the like; extracellular fluids inareas immediately outside of cells; in vesicles; in vascular tissue ofplants and animals; in biological fluids such as blood, saliva, andurine; in biological fermentation media; in environmental samples suchas water, soil, waste water and sea water, in industrial samples such aspharmaceuticals, foodstuffs and beverages; and in chemical reactors.Detection and quantitation of the target ion in a sample can helpcharacterize the identity of an unknown sample, or facilitate qualitycontrol of a sample of known origin.

In one embodiment of the invention, the sample contains cells, and theindicator is combined with the sample in such a way that the indicatoris present within the sample cells. By selection of the appropriatechelating moiety, fluorophore, and the substituents thereon, indicatorsare prepared that will selectively localize in desired organelles, andprovide measurements of the target ion in those organelles. Conjugatesof the indicators of the invention with organelle-targeting peptides areused to localize the indicator to the selected organelle, facilitatingmeasurement of target ion presence or concentration within the organelle(as described in U.S. Pat. No. 5,773,227). Alternatively, selection of alipophilic fluorophore, or a fluorophore having predominantly lipophilicsubstituents will result in localization in lipophilic environments inthe cell, such as cell membranes. Selection of cationic indicators willtypically result in localization of the indicator in mitochondria.

In another aspect of the invention, a composition of matter comprisesany of the compounds described above, and optionally includes a metalion. In one embodiment, the compounds of the invention, in any of theembodiments described above, are associated, either covalently ornoncovalently, with a surface such as a microfluidic chip, a siliconchip, a microscope slide, a microplate well, or another solid matrix,and is combined with the sample of interest as it flows over thesurface. The detectable optical response is therefore detected on thematrix surface itself, typically by use of an instrumental. Thisembodiment of the invention is particularly suited to high-throughputscreening using automated methods.

Quantification of target ion levels in samples is typically accomplishedusing the indicators of the present invention by methods known in theart. For example, the ratiometric measurement of ion concentrationprovides accurate measurement of ion concentrations by the treatment ofthe fluorescence data as the ratio of excitation or fluorescenceintensities at two wavelengths, rather than the absolute intensity at asingle wavelength. Using the ratio method, a number of variables thatmay perturb the ion concentration measurements are eliminated. Inparticular, ion-dependent factors that affect the signal intensity, suchas nonuniform intracellular dye concentrations, probe leakage, dyebleaching and cell thickness, are canceled in the ratio measurements,since these parameters have a similar effect on intensities at bothwavelengths. While the ratio method can be used to determineconcentrations using observation of either the excitation spectra of theindicator, the emission spectra of the indicator, or both, in the caseof the indicators of the present invention, the shift in excitationenergy upon binding metal ions makes observation of the excitationspectrum a more useful technique. In either case, to achieve maximalutility, the indicator must be calibrated (to compensate for variance inthe dissociation constant of the indicator due to ionic strength,viscosity, or other conditions within the sample). To calibrate theindicator, ionophores such as A-23187, gramicidin, valinomycin, orionomycin are used. Non-ratiometric analysis can also be accomplished bycalibration with a second fluorescent dye present in the sample.

The optical response of the indicator to the ion can be detected byvarious means that include measuring absorbance or fluorescence changeswith an instrument, visually, or by use of a fluorescence sensingdevice. Several examples of fluorescence sensing devices are known, suchas fluorometers, fluorescence microscopes, laser scanners, flowcytometers, and microfluidic devices, as well as by cameras and otherimaging equipment. These measurements may be made remotely byincorporation of the fluorescent ion sensor as part of a fiber opticprobe. The indicator is covalently attached to the fiber optic probematerial, typically glass or functionalized glass (e.g., aminopropylglass) or the indicator is attached to the fiber optic probe via anintermediate polymer, such as polyacrylamide. The indicator solution isalternatively incorporated non-covalently within a fiber optic probe, aslong as there is a means whereby the target ion can come into contactwith the indicator solution.

C. Kits of the Invention

Due to the advantageous properties and the simplicity of use of theinstant crown ether compounds, they are particularly useful in theformulation of a kit for the complexation, detection, quantification ormonitoring of selected target ions, comprising one or more compounds orcompositions of the invention in any of the embodiments described above(optionally in a stock solution), instructions for the use of the crownether compound to complex or detect a desired target ion, and optionallycomprising additional components. In one aspect, the compounds of theinvention are associated with a surface, such as a chip, microplatewell, or other solid matrix, and the sample of interest flows over thesurface. The detectable optical response is therefore detected on thematrix surface itself.

Therefore a kit of the present invention for binding a target metal ionin a sample comprises a compound represented by Formula (I) or Formula(II) and comprising one or more components selected from the groupconsisting of a calibration standard of a metal ion, an ionophore, afluorescent standard, an aqueous buffer solution and an organic solvent.

The additional kit components may be selected from, without limitation,calibration standards of a target ion, ionophores, fluorescencestandards, aqueous buffers, and organic solvents.

The additional kit components are present as pure compositions, or asaqueous solutions that incorporate one or more additional kitcomponents. Any or all of the kit components optionally further comprisebuffers.

The examples below are given so as to illustrate the practice of thisinvention. They are not intended to limit or define the entire scope ofthis invention.

EXAMPLES Example 1 Preparation of a Tetraaza-crown Ether

2-Nitroaniline is acylated with diglycolyl chloride in anhydrous THF,using 0.5 equivalents of diglycolyl chloride in dilute solution. A 0.1 MTHF solution of diglycolyl chloride is slowly added to a 0.1 M THFsolution of 2-nitroaniline, containing two equivalents of triethylamine.After TLC indicates consumption of all starting 2-nitroaniline,volatiles are removed in vacuo and the residue is partitioned betweenwater and ethyl acetate. The ethyl acetate layer is concentrated to giveCompound 1.

Compound 1 is dissolved in methanol and treated with 20% by weight of10% palladium on carbon. The resulting mixture is shaken under 40 psihydrogen until analysis by thin layer chromatography (TLC) indicates thereaction is complete. The reaction mixture is filtered and thebis-aniline 2 is isolated by concentration in vacuo of the filtrate.

Compound 2 is dissolved in DMF to achieve a 0.1 M solution and thentreated with 0.9 equivalents of 1,2-dibromoethane, two equivalents ofdilsopropylethylamine, and catalytic sodium iodide. The resultingsolution is heated until most of compound 2 is consumed, as judged byTLC analysis. The volatiles are removed in vacuo, and the residue ispartitioned between ethyl acetate and water. The ethyl acetate layer isconcentrated in vacuo to give compound 3.

Compound 3 is dissolved in acetonitrile to achieve a 0.5 Mconcentration. Ten equivalents of formaldehyde, as a 37% aqueoussolution, are added followed by 4 equivalents of sodiumcyanoborohydride. The pH of the reaction mixture is adjusted to 7 withacetic acid, and the resulting solution is stirred until TLC analysisindicates consumption of compound 3 and formation of compound 4.Volatiles are removed in vacuo, and the residue is partitioned betweenethyl acetate and water. The ethyl acetate layer is concentrated invacuo to give compound 4.

Compound 4 is dissolved in THF at 1 M concentration, and treated with 10equivalents of borane-THF (1M). The resulting solution is heated atreflux until compound 4 is consumed and compound 5 is formed, as judgedby TLC analysis. The reaction is quenched by addition of 5% aqueoussodium hydroxide, followed by extraction with ethyl acetate. The ethylacetate layer is concentrated in vacuo to give compound 5.

Compound 5 is dissolved in DMF at 0.5 M concentration. Ten equivalentsof methyl bromoacetate and 4 equivalents of diisopropylethylamine areadded, and the resulting solution is stirred at 100 degrees centigradeuntil TLC analysis indicates consumption of compound 5 and formation ofcompound 6. Volatiles are removed in vacuo, and the residue ispartitioned between water and ethyl acetate. The ethyl acetate layer isconcentrated in vacuo to give compound 6, which can be further purifiedby flash chromatography on silica gel using methanol in chloroform aseluant.

Example 2 Preparation of (2′-Nitrophenoxy)-2-chloroethane (7)

A suspension of 2-nitrophenol (50.0 g, 0.36 mol), 1-bromo-2-chloroethane(45 mL, 0.54 mol), and K₂CO₃ (100.0 g, 0.72 mol) in DMF (200 mL) wasstirred at 90° C. for 2 h, cooled to room temperature, poured intoice-water, filtered, washed with H₂O and dried to give Compound 7, 70.3g (96%) as a yellow solid.

Example 3 Preparation of1-(5′-methyl-2′-nitrophenoxy)-2-(2″-nitrophenoxy)ethane (8)

A suspension of Compound 7 (60.45 g, 0.30 mol), 5-methyl-2-nitrophenol(50.49 g, 0.33 mol), and K₂CO₃(82.80 g, 0.60 mol) in DMF (450 mL) wasstirred at 130° C. for 18 h, cooled to room temperature, poured intoice-water, filtered, washed with H₂O and dried to give Compound 8, 89.50g (93.5%) as an orange solid.

Example 4 Preparation of1-(2′-amino-5′-methylphenoxy)-2-(2″-aminophenoxy)ethane (9)

Compound 8 (24.0 g, 75 mmol) was hydrogenated over 10% Pd/C (1.5 g) inDMF (250 mL) at 40 psi for 18 h. The mixture was filtered from catalystthrough a CELITE pad on a fritted glass filter. The filtrate wasevaporated and ether (50 mL) was added. The product was filtered, washedwith ether and dried to give compound 9, 18.0 g (95%) as a brown solid.

Example 5 Preparation of Compound 10

To a stirred mixture of diamine Compound 9 (13.10 g, 50 mmol) and Et₃N(20.8 mL, 150 mmol) in dry THF (2.5 L) was slowly added over 12 h asolution of diglycolyl dichloride (6.3 mL, 55 mmol) in dry THF (0.5 L).The reaction mixture was stirred for 6 h, filtered from the precipitatedhydrochloride, and washed with THF. The combined organic filtrate wasevaporated. The residue was dissolved in CHCl₃ (800 mL) and washedsuccessively with 0.5 M HCl, H₂O, saturated NaHCO₃ then saturated NaCl.The organic layer was dried over MgSO₄ and evaporated. Ether (100 mL)was added and the precipitated product was filtered, washed with etherand dried to give Compound 10, 16.0 g (90%) as an off-white solid.

Example 6 Preparation of Compound 11

To a suspension of diamide Compound 10 (15.60 g, 44 mmol) in dry THF(400 mL) was added a 1 M solution of BH₃-THF complex in dry THF (390 mL,390 mmol). The mixture was stirred at 70° C. for 16 h. Dry methanol (300mL) was added dropwise over 1 h into a boiling mixture with strong gasevaluation. The mixture was heated at reflux for 2 h, cooled to roomtemperature, evaporated and co-evaporated with MeOH to destroy theborane complex. Ether (100 mL) was added. The solid product wasfiltered, washed with ether and dried to give Compound 11, 12.3 g (85%)as an off-white solid.

Example 7 Preparation of Compound 12

A mixture of diamine 11 (6.56 g, 20 mmol), methyl bromoacetate (38 mL,400 mmol), diisopropylethylamine (DIPEA) (104 mL, 600 mmol), and Nal(1.50 g, 10 mmol) in MeCN (300 mL) was heated at reflux for 70 h. Aftercooling to room temperature, the MeCN was evaporated. The residue wasdissolved in CHCl₃, washed with 1% AcOH then H₂O, dried and evaporated.The residual oily product was cooled to 0° C. and washed with coldhexane to remove most of the alkylating reagent. The crude product,compound 12, was used immediately in the next step without furtherpurification.

Example 8 Preparation of Compound 13

To a stirred solution of the Vilsmeier reagent prepared from POCl₃ (4.65mL, 50 mmol) and 30 mL DMF was added over 5 min a solution of Compound12 (4.8 g, 10 mmol) in DMF (20 mL). The mixture was stirred for 16 h,cooled by the addition of ice and neutralized with saturated K₂CO₃ to pH7-8. The suspension was extracted with CHCl₃, washed successively with0.1 M HCl, saturated NaCl, saturated NaHCO₃ and saturated NaCl and thenevaporated. The residue was dissolved in CHCl₃ and chromatographed onSiO₂ with an EtOAc gradient in hexanes (20% to 30%) to give aldehyde 13,2.60 g (52%) as colorless crystals.

Example 9 Preparation of Compound 14

Compound 14 was prepared analogously with Compound 13, starting with5-methyl-2-nitrophenol in place of 2-nitrophenol, and utilizing4-benzyloxy-2-nitrophenol in place of 5-methyl-2-nitrophenol.

Example 10 Preparation of Compound 15

Compound 14 (3.06 g, 5.0 mmol) was hydrogenated over 10% Pd/C (0.50 g)in AcOH (100 mL) at 50 psi for 5 h. The mixture was filtered through aCELITE pad on a fritted glass filter. The filtrate was evaporated, ether(50 mL) was added, the product was filtered, washed with ether and driedto give Compound 15, 2.13 g (82%) as a colorless solid.

Example 11 Preparation of Compound 16

To a suspension of compound 15 (0.800 g, 1.55 mmol), K₂CO₃ (1.04 g, 7.5mmol) and Nal (0.03 g, 0.2 mmol, catalyst) in DMF (10 mL) was addeddropwise t-butyl bromoacetate (0.45 mL, 3.0 mmol). The mixture wasstirred for 16 h, diluted with H₂O, extracted with CHCl₃, dried overMgSO₄ and evaporated. The residue was dissolved in hexane: EtOAc (1:1)and chromatographed on silica gel with 40% EtOAc in hexanes to givealdehyde 16, 0.875 g (90%) as colorless crystals.

Example 12 Preparation of Compound 17

A stirred mixture of diamine 11 (3.38 g, 10 mmol), bromoacetonitrile (14mL, 200 mmol), DIPEA (26 mL, 150 mmol), Nal (1.5 g, 10 mmol; catalyst)in MeCN (150 mL) was heated at reflux for 70 h. After cooling to roomtemperature the MeCN was evaporated. The residue was dissolved in CHCl₃(800 mL), washed with H₂O, dried over MgSO₄ and evaporated. The residuewas chromatographed on SiO₂ with 40% EtOAc in hexanes to give Compound17, 3.56 g (90%) as a colorless solid.

Example 13 Preparation of Compound 18

To a stirred solution of the Vilsmeier reagent prepared from POCl₃ (9.5mL, 102 mmol) and 60 mL DMF was added over 5 min to a solution ofCompound 17. The mixture was stirred for 100 h at 40° C., cooled by theaddition of ice and neutralized with saturated K₂CO₃ to pH 8. Thesuspension was extracted with CHCl₃, washed with H₂O, dried over MgSO₄and evaporated. The residue was chromatographed over SiO₂ using CHCl₃ aseluant to give compound 18, 3.12 g (73%) as colorless crystals.

Example 14 Preparation of Compound 19

A mixture of aldehyde 18 (3.08 g, 7.3 mmol), ethylene glycol (8 mL, 140mmol), p-toluenesulphonic acid (0.20 g, catalyst) and benzene (80 mL)was refluxed with a Dean-Stark trap for 4 h. After cooling to roomtemperature the mixture was evaporated. The residue was dissolved inCHCl₃ (300 mL), washed with saturated NaHCO₃, dried over MgSO₄, andevaporated to give compound 19, 2.82 g (85%) as an orange solid.Compound 19 was pure on TLC and used in the next step without additionalpurification.

Example 15 Preparation of Compound 20

To a solution of Compound 19 (0.200 g, 0.44 mmol) in MeOH (25 mL) and 1M NaOH (15 mL, 15 mmol) was added over 5 min 30% H₂O₂ (5 mL, catalyst).The mixture was stirred for 1 h, then acidified with 1 M HCl to pH 1.The acidified mixture was stirred for 30 min, diluted with 3 M NaOAc(150 mL), extracted with CHCl₃, washed with saturated NaHCO₃, dried overMgSO₄ and evaporated to give aldehyde 20, 0.191 g (95%) as a colorlesscrystalline material, pure by TLC.

Example 16 Preparation of Compound 21

A mixture of aldehyde 13 (1.150 g, 2.3 mmol) and 4-fluororesorcinol(0.670 g, 5.2 mmol) in MeSO₃H (25 mL) was stirred overnight and thenpoured into 3 M NaOAc (300 mL). The precipitated solid was filtered,washed with H₂O and dried to give compound 21, 1.605 g (97%) as anoff-white solid. Compound 21 was unstable to oxidation and was used inthe next step without additional purification.

Example 17 Preparation of Compound 22

A mixture of Compound 21 (150 mg, 0.21 mmol) and chloranil (246 mg, 1.0mmol) in MeOH (5 mL) and CHCl₃ (5 mL) was refluxed for 50 h, then cooledto room temperature, filtered from excess oxidizer, and evaporated. Theresidue was purified by preparative TLC on two SiO₂ plates using CHCl₃:MeOH:AcOH (20:2:1) to give compound 22, 44 mg (20%) as a red-brownsolid.

Example 18 Preparation of Compound 23

A mixture of Compound 22 (30 mg, 0.04 mmol) in MeOH (5 mL) and 1 M KOH(1 mL, 1.0 mmol) was stirred for 16 h, then neutralized with 1 M HCl topH 7, and evaporated to dryness. The residue was purified by preparativeTLC on two SiO₂ plates using CHCl₃: MeOH:AcOH (13:3:1) to give compound23, 21 mg (74%) as a brown solid.

Example 19 Preparation of Compound 24

A mixture of Compound 22 (78 mg, 0.1 mmol), bromomethyl acetate (0.05mL, 0.5 mmol), and DIPEA (0.175 mL, 1 mmol) in DMF (1 mL) was stirredfor 2 h, then poured into 1% AcOH (200 mL). The suspension was extractedwith CHCl₃, washed with H₂O, filtered and evaporated. The residue waspurified by preparative TLC on two SiO₂ plates using 5% MeOH in CHCl₃:to give compound 24, 25 mg (32%) as a brown solid.

Example 20 Preparation of Compound 25

A mixture of Compound 23 (69 mg, 0.1 mmol), bromomethyl acetate (0.05mL, 0.5 mmol), and DIPEA (0.175 mL, 1 mmol) in DMF (1 mL) was stirredfor 2 h, then poured into 1% AcOH (200 mL). The suspension was extractedwith CHCl₃, washed with H₂O, filtered and evaporated. The residue waspurified by preparative TLC on two SlO₂ plates using 5% MeOH in CHCl₃ togive Compound 25 as a brown solid.

Example 21 Preparation of Compound 26

A mixture of aldehyde 13 (250 mg, 0.5 mmol), 3-dimethylaminophenol (157mg, 1.1 mmol), and p-toluenesulphonic acid (10 mg, catalyst) inpropionic acid (5 mL) was stirred overnight at 60° C., then cooled toroom temperature and poured into 3 M NaOAc (100 mL). The precipitatedsolid was filtered, washed with H₂O and dried to give compound 26, 360mg (97%) as a rose-colored solid. Compound 26 was unstable to oxidationand was used in subsequent reactions without additional purification.

Example 22 Preparation of Compound 27

A mixture of Compound 26 (660 mg, 0.81 mmol) and chloranil (400 mg, 1.62mmol) in MeOH (25 mL) and CHCl₃ (25 mL) was stirred for 2 h, filteredfrom excess oxidizer, and evaporated. The residue was purified bychromatography on SiO₂ using 9% MeOH and 1% AcOH in CHCl₃ as eluant togive the crude product, which was again chromatographed on SiO₂ usingthe same eluant to give Compound 27, 112 mg (19%) as crimson solid.

Example 23 Preparation of Compound 28

A mixture of Compound 27 (40 mg, 0.05 mmol) in MeOH (2 mL) and 1 M KOH(1 mL, 1.0 mmol) was stirred for 16 h and then evaporated to dryness.The residue was purified by chromatography on a SEPHADEX LH-20 columnusing H₂O as eluant to give salt 28, 8 mg (21%) as red flakes afterlyophilization.

Example 24 Preparation of Compound 29

A mixture of Compound 28 (75 mg, 0.1 mmol), bromomethyl acetate (0.1 mL,1.0 mmol), and DIPEA (0.35 mL, 2 mmol) in DMF (2 mL) was stirred for 2h, then poured into 1% AcOH (200 mL). The suspension is extracted withCHCl₃, washed with H₂O, filtered and evaporated. The residue waspurified by preparative TLC on two SiO₂ plates using 5% MeOH in CHCl₃ togive Compound 29 as a red solid.

Example 25 Preparation of Compound 30

Compound 30 was prepared analogously to Compound 27, using8-hydroxyjulolidine rather than m-dimethylaminophenol.

Example 26 Preparation of Compound 31

Compound 31 was prepared form Compound 27 using the procedure forpreparing Compound 28 from Compound 27.

Example 27 Preparation of Compound 32

To a solution of aldehyde 13 (250 mg, 0.5 mmol) in CH₂Cl₂ (20 mL) wasadded 2,4-dimethylpyrrole (0.125 mL, 1.2 mmol). The solution was stirredfor 5 min, then TFA (0.046 mL, 0.6 mmol) is introduced. After 16 h, themixture was diluted with CHCl₃ (150 mL), washed with 2%tetrabutylammonium hydroxide (150 mL) then H₂O then evaporated. Theresidue was dissolved in toluene (20 mL) and stirred for 3 h withchloranil (148 mg, 0.6 mmol). DIPEA (0.87 mL, 5 mmol) was introduced,followed by BF₃ etherate (0.52 mL, 4 mmol). The mixture was evaporatedand the residue was purified by chromatography on SiO₂ using a gradientof 0-2% MeOH in CHCl₃ to give compound 32, 102 mg (28%) as a brownsolid.

Example 28 Preparation of Compound 33

A mixture of aldehyde 13 (1.00 g, 2.0 mmol), the Wittig base(4methoxycarbonyl-2-nitrobenzyl)triphenylphosphonium bromide (1.34 g,2.5 mmol), and K₂CO₃ (1.38 g, 10 mmol) in DMF (20 mL) was stirred for 16h at 95° C. More of the Wittig base (0.500 g, 0.93 mmol) was added andthe mixture was stirred for an additional 6 h, then cooled to roomtemperature and poured into H₂O. The solution was acidified with 1 M HClto pH 3, extracted with CHCl₃, dried over MgSO₄, and evaporated. Thecrude product was purified by chromatography on SiO₂ using CHCl₃ then 2%MeOH in CHCl₃ to give compound 33, 0.915 g (68%) as an orange solid.

Example 29 Preparation of Compound 34

A mixture of the ethylene derivative 33 (70 mg, 0.1 mmol) andtriethylphosphite (3 mL) was heated at 120° C. for 6 h, then evaporatedand subsequently co-evaporated with DMF (3×10 mL). The residue waspurified by preparative TLC on two SiO₂ plates using 7% MeOH in CHCl₃ aseluant to give compound 34, 52 mg (80%) as a slightly yellowish solid.

Example 30 Preparation of Compound 35

A mixture of Compound 34 (300 mg, 0.46 mmol), and 1 M KOH (6 mL, 6 mmol)in MeOH (25 mL) was stirred for 16 h. More 1 M KOH (5 mL, 5 mmol) wasadded and the mixture was stirred for an additional 6 h, then evaporatedto half its original volume, and acidified with 1 M HCl to pH 3. Thesuspension was extracted with CHCl₃ then with n-BuOH. The combinedorganic extract was filtered and was evaporated. Ether (20 mL) was addedand the precipitated solid was filtered and washed with ether to givecompound 35, 157 mg (57%) as an off-white solid.

Example 31 Preparation of Compound 36

A mixture of Compound 35 (72 mg, 0.12 mmol), bromomethyl acetate (0.14mL, 1.0 mmol), and DIPEA (0.35 mL, 2 mmol) in DMF (5 mL) was stirred for16 h, poured into 1% AcOH (200 mL), extracted with CHCl₃, washed withH₂O then evaporated. The residue was purified by preparative TLC on twoSiO₂ plates using 5% MeOH in CHCl₃ to give Compound 36, 3 mg (3%) as abrown solid.

Example 32 Preparation of Compound 37

A mixture of aldehyde 16 (630 mg, 1.0 mmol), 3-dimethylaminophenol (330mg, 2.4 mmol), and p-toluenesulphonic acid (20 mg, catalyst) inpropionic acid (5 mL) was stirred for 16 h at 60° C., then cooled toroom temperature and poured into 3 M NaOAc (150 mL). The precipitatedsolid was filtered, washed with H₂O and dried to give Compound 37, 360mg (97%) as a rose solid. Compound 37 is unstable to oxidation and wasused in next step without additional purification.

Example 33 Preparation of Compound 38

A mixture of Compound 37 (2.50 g, 2.9 mmol) and chloranil (1.42 g, 5.8mmol) in MeOH (100 mL) and CHCl₃ (100 mL) was stirred for 4 h, filteredfrom excess oxidizer, and evaporated. The residue was purified bychromatography on SiO₂ using a gradient of 6-10% MeOH and 1% AcOH inCHCl₃ to give compound 38, 1.13 g (45%) as a crimson solid.

Example 34 Preparation of Compound 39

A mixture of Compound 38 (500 mg, 0.58 mmol) in CH₂Cl₂ (20 mL) and TFA(20 mL) was stirred for 4 h, then evaporated and co-evaporated withCHCl₃. Ether (25 mL) was added to the residue and the precipitatedproduct was filtered and washed with ether to give Compound 39, 447 mg(95%) as a violet-red solid.

Example 35 Preparation of Compound 40

A mixture of Compound 39 (20 mg, 0.1 mmol), bromomethyl acetate (0.025mL, 0.25 mmol), and DIPEA (0.08 mL, 0.5 mmol) in DMF (1 mL) was stirredfor 3 h. More bromomethyl acetate (0.025 mL, 0.25 mmol) and DIPEA (0.08mL, 0.5 mmol) were added and the mixture was stirred for an additional 3h, diluted with CHCl₃ (100 mL), washed with 1% AcOH then H₂O thenevaporated. The residue was purified by preparative TLC on two SiO₂plates using 10% MeOH and 0.5% AcOH in CHCl₃ to give Compound 40, 12 mg(54%) as a dark red solid.

Example 36 Preparation of Compound 41

To a stirred solution of Compound 39 (162 mg, 0.2 mmol) and pyridine(0.08 mL, 1 mmol) in DMF (5 mL), was added dryN-trifluoroacetoxysuccinimide (90 mg, 0.4 mmol). After 7 h moreN-trifluoroacetoxysuccinimide (90 mg, 0.4 mmol) and pyridine (0.08 mL, 1mmol) were added, and the mixture was stirred for 16 h more. AnalyticalTLC confirmed the formation of the single product, while startingmaterial 39 was consumed. Compound 41 was very reactive and unstabletowards isolation attempts. It was used in further transformations uponpreparation in DMF solution without isolation and purification.

Example 37 Preparation of Compound 42

To a stirred solution of succinimidyl ester 41 prepared as in Example 36from compound 39 (81 mg, 0.1 mmol) in DMF (2 mL) was added dryhexadecylamine (135 mg, 0.5 mmol). The mixture was stirred for 16 h,diluted with CHCl₃ (150 mL), washed with 1% AcOH the H₂O, dried overMgSO₄, and evaporated. The residue was purified by preparative TLC ontwo SiO₂ plates using CHCl₃ as eluant to give Compound 42, 57 mg (54%)as a red oil.

Example 38 Preparation of Compound 43

To a stirred solution of succinimidyl ester 41 prepared as in Example 36from Compound 39 (81 mg, 0.1 mmol) in DMF (2 mL) was quickly introduceda solution of 5-aminopentanoic acid (40 mg, 0.3 mmol) and 1 M methanolictetrabutylammonium hydroxide (1 mL, 1 mmol) in H₂O (5 mL). The mixturewas stirred for 4 h, then diluted with H₂O (100 mL), acidified with AcOHto pH 4, extracted with CHCl₃, dried over MgSO₄ and evaporated. Theresidue was purified by preparative TLC on two SiO₂ plates using 10%MeOH and 2.5% AcOH in CHCl₃ as the eluant to give Compound 43, 56 mg(61%) as a red solid.

Example 39 Preparation of Compound 44

To a stirred solution of Compound 43 (37 mg, 0.04 mmol) and pyridine(0.08 mL, 1 mmol) in DMF (1 mL), was added dryN-trifluoroacetoxysuccinimide (36 mg, 0.16 mmol). After 4 h, moreN-trifluoroacetoxysuccinimide (36 mg, 0.4 mmol) and pyridine (0.08 mL, 1mmol) are added, and the mixture was stirred for an additional 16 h. Themixture was diluted with CHCl₃ (100 mL), washed with 1% AcOH then H₂Othen evaporated. Hexanes (5 mL) were added to the residue. Theprecipitated solid was filtered, washed with hexanes and dried to giveCompound 44, 15 mg (37%) as a dark red solid.

Example 40 Preparation of Compound 45

A solution of succinimidyl ester 41 (81 mg, 0.1 mmol) in DMF (2 mL) wasadded to 5 mL of aqueous aminodextran (100 mg, 0.037 eq.) and 1 Mmethanolic tetrabutylammonium hydroxide (1 mL, 1 mmol). The mixture wasstirred for 16 h, poured into MeOH (400 mL), and the precipitatedconjugate was filtered off and washed with MeOH. The crude product wasdissolved in H₂O (3 mL), filtered through a membrane filter and loadedonto a SEPHADEX G-15 resin column pre-equilibrated with H₂O. The coloredfraction was eluted in a void volume and lyophylized to give labeleddextran 45, (41 mg) as a red solid.

Example 41 Preparation of Compound 46

To a stirred solution of succinimidyl ester 41 (81 mg, 0.1 mmol) in DMF(2 mL) was added Et₃N (0.14 mL, 1 mmol) followed by2-(4-aminophenyl)ethylamine (0.05 mL, 0.5 mmol). The mixture was stirredfor 2 h, diluted with CHCl₃ (150 mL), washed with 1% AcOH then H₂O thenevaporated. Ether (5 mL) was added to the residue, and the solid wasfiltered off, washed with ether and dried to give Compound 46 as a darkred solid, pure on TLC and HPLC.

Example 42 Preparation of Compound 47

To a solution of Compound 46 (9 mg, 0.01 mmol) in AcOH (2 mL) was addeda 1 M CSCl₂ solution in CHCl₃ (0.1 mL, 0.1 mmol). The mixture wasstirred for 2 h then evaporated. Ether (5 mL) was added to the residue,and the precipitated solid was filtered off, washed with ether and driedto give Compound 47, 9 mg (95%) as a dark red solid. Compound 47 reactsquickly with n-BuNH₂ in pre-column derivatization for HPLC analysis.

Example 43 Preparation of Compound 48

To a stirred solution of aldehyde 16 (630 mg, 1.0 mmol) in CH₂Cl₂ (40mL), 2,4-dimethylpyrrole (0.25 mL, 2.4 mmol) was added. The solution wasstirred for 5 min, then TFA (0.1 mL, 1.2 mmol) was introduced. Themixture was stirred for 16 h, and diluted with CHCl₃ (300 mL). Thechloroform solution was washed with 2% tetrabutylammonium hydroxide thenH₂O, evaporated, and subsequently co-evaporated with toluene. Theresidue was dissolved in toluene (40 mL), stirred for 2 h with chloranil(296 mg, 1.2 mmol), then DIPEA (1.74 mL, 10 mmol) was introduced,followed by BF₃ etherate (1.04 mL, 8 mmol). The mixture was evaporatedand the residue was purified by chromatography on SiO₂ using a gradientof 0-1% MeOH in CHCl₃ to give Compound 48, 280 mg (35%) as a brownsolid.

Example 44 Preparation of Compound 49

A mixture of Compound 48 (120 mg, 0.15 mmol) in CHCl₃ (6 mL) and TFA(0.4 mL) was stirred for 16 h, then evaporated and co-evaporated withCHCl₃. The residue was purified by preparative TLC on two SiO₂ platesusing 10% MeOH in CHCl₃ as eluant to give Compound 49, 82 mg (77%) as abrown solid.

Example 45 Preparation of Compound 50

A mixture of Compound 49 (80 mg, 0.11 mmol), bromomethyl acetate (0.05mL, 0.5 mmol), and DIPEA (0.17 mL, 1.0 mmol) in DMF (2 mL) was stirredfor 2 h, poured into H₂O (150 mL), and extracted with CHCl₃. The extractwas dried over MgSO₄ then evaporated. The residue was purified bypreparative TLC on two SiO₂ plates using 3% MeOH and 0.5% AcOH in CHCl₃to give Compound 50, 46 mg (52%) as a brown solid.

Example 46 Preparation of Compound 51

A mixture of aldehyde 15 (800 mg, 1.55 mmol),5-carboxy-2-chloromethyloxazole (300 mg, 1.86 mmol), K₂CO₃ (1.07 g, 7.75mmol), and Nal (75 mg, 0.5 mmol; catalyst) in DMF (20 mL) was stirred at135° C. for 4 h, then poured into H₂O. The mixture was acidified with 1M HCl to pH 3, extracted with CHCl₃, dried over MgSO₄ then evaporated.The residue was purified by chromatography on SiO₂ using a gradient of5-20% MeOH and 1-2% AcOH in CHCl₃ as eluant to give Compound 51, 255 mg(26%) as a yellowish solid.

Example 47 Preparation of Compound 52

A mixture of Compound 51 (62 mg, 0.1 mmol), bromomethyl acetate (0.05mL, 0.5 mmol), and DIPEA (0.17 mL, 1.0 mmol) in DMF (1 mL) was stirredfor 2 h, poured into 1% AcOH (100 mL), and extracted with CHCl₃. Then 3M NaOAc (100 mL) was added to the aqueous phase and the mixture wasextracted with more CHCl₃. The combined extracts were dried over MgSO₄then evaporated. The residue was purified by preparative TLC on two SiO₂plates using 5% MeOH in CHCl₃ to give Compound 52, 29 mg (42%) as anorange solid.

Example 48 Preparation of Compound 53

A mixture of aldehyde 15 (1.40 g, 2.71 mmol),2-chloromethyl-5-ethoxycarbonyloxazole (0.564 g, 1.86 mmol), K₂CO₃ (1.87g, 13.60 mmol), and Nal (0.150 g, 1.00 mmol; catalyst) in DMF (30 mL)was stirred at 135° C. for 4 h and then poured into H₂O. The mixture wasacidified with 1 M HCl to pH 3, extracted with CHCl₃ and evaporated. Theresidue was purified by chromatography on SiO₂ using 1% MeOH in CHCl₃ aseluant to give Compound 53, 1.260 g (71%) as a yellowish solid.

Example 49 Preparation of Compound 54

A mixture of Compound 53 (376 mg, 0.5 mmol) and 1 M KOH (3 mL, 3.0 mmol)in MeOH (5 mL) was stirred for 5 h, then evaporated to 1/5 volume, andacidified with 1 M HCl to pH 2.8. The precipitated solid was filtered,washed with H₂O and dried on a filter. This crude product was suspendedin H₂O (2 mL) and then made basic with 0.1 M KOH to pH 9.5. The solutionwas loaded onto a SEPHADEX LH-20 resin column and chromatographed usingH₂O as eluant to give Compound 54, 256 mg (72%) as a yellow-greenishsolid.

Example 50 Preparation of Compound 55

A mixture of Compound 54 (71 mg, 0.1 mmol), bromomethyl acetate (0.1 mL,1.0 mmol), and DIPEA (0.35 mL, 2.0 mmol) in DMF (2 mL) was stirred for 4h, diluted with CHCl₃, then washed with 1% AcOH then H₂O and evaporated.The residue was purified by preparative TLC on two SiO₂ plates using 5%MeOH and 0.5% AcOH in CHCl₃ to give compound 55, 63 mg (77%) as anorange solid.

Example 51 Preparation of Compound 56

A mixture of aldehyde 16 (315 mg, 0.5 mmol), the Wittig base(4-methoxycarbonyl-2-nitrobenzyl)triphenylphosphonium bromide (375 mg,0.7 mmol), and K₂CO₃ (345 mg, 2.5 mmol) in DMF (3 mL) was stirred for 6h at 95° C. then cooled to room temperature and poured into H₂O. Thesolution was acidified to pH 5 with 1 M HCl and extracted with CHCl₃.The extract was dried over MgSO4 and evaporated. The crude product waspurified by chromatography on SlO₂ using CHCl₃, then 0.5% MeOH in CHCl₃to give Compound 56, 339 mg (84%) as an orange solid.

Example 52 Preparation of Compound 57

A mixture of the ethylene derivative 56 (338 mg, 0.42 mmol) andtriethylphosphite (8 mL) was heated at 130° C. for 7 h, then evaporatedand subsequently co-evaporated with DMF. The residue was first purifiedby chromatography on SiO₂ using CHCl₃ followed by 1% MeOH in CHCl₃, thenby preparative TLC on two SiO₂ plates using 50% EtOAc in hexanes aseluant to give Compound 57, 112 mg (34%) as a slightly yellowish solid.

Example 53 Preparation of Compound 58

A mixture of Compound 57 (104 mg, 0.13 mmol) in CH₂Cl₂ (2 mL) and TFA (2mL) was stirred for 3 h, then evaporated and co-evaporated with CHCl₃.The residue was purified by preparative TLC on two SiO₂ plates using 7%MeOH and 2% AcOH in CHCl₃ to give Compound 58, 55 mg (57%) as ayellowish solid.

Example 54 Preparation of Compound 59

A mixture of Compound 58 (22 mg, 0.03 mmol), bromomethyl acetate (0.1mL, 1.0 mmol), and DIPEA (0.26 mL, 1.5 mmol) in DMF (2 mL) was stirredfor 16 h, then diluted with CHCl₃ (100 mL), washed with 1% AcOH then H₂Othen evaporated. The residue was purified by preparative TLC on two SiO₂plates using 5% MeOH in CHCl₃ to give compound 59, 16 mg (67%) as anorange solid.

Example 55 Preparation of Compound 60

A mixture of Compound 38 (87 mg, 0.1 mmol) and 1 M KOH (2 mL, 2 mmol) inMeOH (5 mL) was stirred for 16 h, and evaporated. The residue waspurified on SEPHADEX LH-20 resin using H₂O as the eluant. The fractionscontaining product were collected and lyophilized to give Compound 60,12 mg (14%) as a crimson solid.

Example 56 Preparation of Compound 61

A mixture of Compound 60 (8 mg, 0.01 mmol), bromomethyl acetate (0.025mL, 0.25 mmol), and DIPEA (0.08 mL, 0.5 mmol) in DMF (1 mL) was stirredfor 16 h, and diluted with CHCl₃ (100 mL). The solution was washed with1% AcOH then H₂O then evaporated. Ether (5 mL) was added to the residue.The precipitated solid was filtered off and washed with ether to giveCompound 61, 5 mg (50%) as a dark red solid. Compound 61 was pure on TLCand HPLC.

Example 57 Preparation of Compound 62

A mixture of aldehyde 18 (217 mg, 0.5 mmol), the Wittig base(4-methoxycarbonyl-2-nitrobenzyl)triphenylphosphonium bromide (536 mg,0.7 mmol), and K₂CO₃ (345 mg, 2.5 mmol) in DMF (3 mL) was stirred for 16h at 95° C., then cooled to room temperature and poured into H₂O. Thesolution was acidified with 1 M HCl to pH 3, extracted with CHCl₃, driedover MgSO₄ and evaporated. The crude product was purified bychromatography on SiO₂ using CHCl₃, as eluant to give Compound 62, 171mg (56%) as an orange solid.

Example 58 Preparation of Compound 63

A mixture of the ethylene derivative 62 (170 mg, 0.28 mmol) andtriethylphosphite (4 mL) was heated at 120° C. for 16 h, then evaporatedand subsequently co-evaporated with DMF. The residue was purified bypreparative TLC on two SiO₂ plates using 3% MeOH in CHCl₃ as eluant,then on two SiO₂ plates using 40% EtOAc in hexanes as eluant to giveCompound 63, 26 mg (16%) as a yellowish solid.

Example 59 Preparation of Compound 64

A mixture of aldehyde 20 (100 mg, 0.21 mmol), 3-dimethylaminophenol (55mg, 0.40 mmol), and p-toluenesulphonic acid (5 mg, catalyst) inpropionic acid (2 mL) was stirred overnight at 60° C., then cooled toroom temperature and poured into 3 M NaOAc (40 mL). Precipitated solidwas filtered, washed with H₂O and dried to give Compound 64, 140 mg(93%) as a rose-colored solid. Compound 64 was unstable to oxidation andwas used in next step without additional purification.

Example 60 Preparation of Compound 65

A mixture of Compound 64 (138 mg, 0.20 mmol) and chloranil (84 mg, 0.34mmol) in MeOH (5 mL) and CHCl₃ (5 mL) was stirred for 4 h, andevaporated. The residue was purified by chromatography on SiO₂ using agradient of 7-9% MeOH and 0.5-1% AcOH in CHCl₃ as eluant to giveCompound 65, 118 mg (86%) as a dark red solid.

Example 61 Preparation of Compound 66

To a 0.5 M solution of arene 12 in sulfuric acid is added one equivalentof potassium nitrate. The resulting solution is stirred until TLCanalysis of reaction aliquots, treated with water and ether, shows fullconversion to nitroarene 66. The reaction mixture is poured into excessaqueous sodium acetate, followed by extraction with ether. The extractis washed with aqueous sodium bicarbonate and brine, dried overmagnesium sulfate and then concentrated to a reddish-brown oil, which ispurified by trituration with ether-hexanes to give Compound 66 as ayellow powder.

Example 62 Preparation of Compound 67

A 0.5 M solution of Compound 66 in methanol is treated with 10%palladium on charcaol, at 10 wt % of Compound 66. The resulting mixtureis shaken under 40 psi hydrogen until TLC analysis shows full conversionto aniline 67. The reaction mixture is filtered through diatomaceousearth, followed by concentration in vacuo. The residue is trituratedwith ether-hexanes to give pure Compound 67 as an off-white powder.

Example 63 Preparation of Compound 68

A mixed anhydride of 6-arboxytetramethylrhodamine is prepared accordingto the procedure given in U.S. Pat. No. 5,453,517 to Kuhn et al. To asolution of the mixed anhydride and one equivalent ofdiisopropylethylamine in anhydrous THF under nitrogen is added slowly a0.3 M solution of Compound 67. The resulting mixture is stirred untilTLC analysis indicates consumption of Compound 67. The reaction mixtureis concentrated in vacuo, and the residue partitioned between chloroformand water. The chloroform layer is washed with brine and dried overmagnesium sulfate, then concentrated in vacuo. The residue is purifiedby flash chromatography on silica gel using increasing amounts ofmethanol in chloroform as eluant to give pure Compound 68 as a redpowder.

Example 64 Preparation of a Tris-aza Crown Ether (69)

To a dilute solution (0.1 M) of diamine 9 and two equivalents of DIPEAin anhydrous THF is slowly added at room temperature a dilute solution(0.1 M) of N,N-bis(chlorocarbonylmethyl)-aniline. After stirringovernight, the volatiles are removed by evaporation, and the residue ispartitioned between 5% hydrochloric acid and ethyl acetate. The organiclayer is washed with brine, dried, and concentrated. The resultingresidue is purified by column chromatography on silica gel usingincreasing amounts of methanol in chloroform to give pure Compound 69.

Example 65 Preparation of Compound 70

The bisamide 69 is reduced to bisaniline 70 as described for Compound11.

Example 66 Preparation of Compound 71

The bisaniline 70 is alkylated to give Compound 71, as described forCompound 12.

Example 67 Preparation of Compound 72

The bisester 71 is formylated to give the aldehyde Compound 72, asdescribed for Compound 13.

Example 68 Preparation of Compound 73

The aldehyde 72 is condensed with two equivalent of3-(dimethylamino)phenol as described for Compound 26 to give Compound73.

Example 69 Preparation of Compound 74

Compound 73 is oxidized with chloranil to give the chloride salt 74 as ared powder, as described for Compound 27.

Example 70 Preparation of a Thia-substituted Crown Ether (75)

A bis-aza crown ether that incorporates a sulfur atom in the crown isprepared using the procedures of Examples 64-69, except that in place ofN,N-bis(chlorocarbonylmethyl)-aniline, the bis-acid chloride ofthiodiglycolic acid is used (Cl(C═O)CH₂—S—CH₂(C═O)Cl). After preparationof the crown ether itself, condensation with3-(N,N-dimethylamino)phenol, and oxidation, the chloride salt of thethia-crown ether, Compound 75, is isolated as a red powder.

Example 71 Determination of Na+, K+, Li+ and Tb+ Indicator FluorescenceResponse

The fluorescence response of a selected compound of the invention as afunction of ion concentration was determined by dissolving a sample ofthe pure compound in 3 mL of each of two solutions: solution 1 (“high”)consists of 200 mM NaCl (or other appropriate ion) and 10 mM MOPS bufferat pH 7.05; solution 2 (“zero”) consists of 10 mM MOPS buffer at pH 7.05in deionized water. A series of curves are generated by cross dilutionbetween the two solutions to arrive at intermediate concentrations ofNa⁺, K+, Li+and Tb+. The emission of a selected indicator in solution 2was scanned and then was repeated to cover the entire range from zero to200 mM Na⁺. For example, the fluorescence emission of the chosenindicator was scanned while the sample was excited at that indicator'sabsorption maximum wavelength, and then 1/100 of the sample was removedand replaced with 1/100 of solution 1 to arrive at a Na⁺ concentrationof 2 mM. This dilution was repeated to cover the entire range from zeroto 1 M Na⁺ and the resulting emission intensities were plotted versusthe ion concentrations. A least-squares fit was used to arrive at theconcentration where the selected indicator was maximally sensitive tochanges in Na⁺ concentration. This corresponded to the dissociationconstant of that indicator for Na⁺ and was expressed as a concentration.

This methodology was repeated for K+, Li+and Tb+ to obtain emissionspectra and to calculate the Kd values. See, Table 2 and FIG. 4

For visible wavelength probes such as Compound 22 (Example 17), Compound27 (Example 22) and Compound 86 (Example 79) the indicator'sfluorescence emission was typically scanned from 450-650 nm while thesample was excited at the absorption maximum wavelength. ForUV-excitable ratiometric probes such as Compound 51 (Example 46), theexcitation wavelengths of the dye in solution 2 were scanned from 260 to450 nm while monitoring constant fluorescence emission at 510 nm (as inFIG. 1).

The binding affinity of a selected indicator for sodium ions, in thepresence of potassium ions, is determined using the process above,except in the presence of 100 mM K⁺ concentrations.

Example 72 Calibration of Sodium Indicator Fluorescence Response inCells

Jurkat cells were loaded with a 5 μM solution of either Compound 25(Example 20), or commercially available SODIUM GREEN tetraacetateindicator (Molecular Probes, Inc., Eugene, Oreg.) and 10 μM for Compound87 (Example 80) for 30 minutes at 37° C. The use of PLURONIC dispersingagent helped dissolve the selected indicator. Intracellular sodiumconcentrations were then established by varying the sodium concentrationof the extracellular buffer in the presence of 2 μM gramicidin (asodium-pore forming antibiotic). The extracellular buffer was set at 0mM, 10 mM, 20 mM, 50 mM, 100 mM, and 145 mM, respectively. Intracellularfluorescence response of the selected indicators was measured using aFACSCAN flow cytometer and associated software, with fluorescenceexcitation at 488 nm. Compound 25 exhibits substantially brighterintracellular fluorescence intensity than the SODIUM GREEN indicator atcomparable Na⁺ concentrations, as shown in FIG. 3. Similarly, asintracellular Na⁺ concentration was increased, Compound 25 exhibits aconsistent increase in fluorescence intensity. Compound 87 demonstratedan increased fluorescent signal with increasing concentrations of sodiumions.

Example 73 Preparation of a Biotinylated Indicator (Compound 76)

To a stirred solution of succinimidyl ester 41 prepared as in Example 36from compound 39 (81 mg, 0.1 mmol) in DMF (2 mL) was added Et₃N (0.27mL, 2 mmol) followed by biotin-cadaverin trifluoracetate (66 mg, 02mmol). The mixture was stirred for 16 h, diluted with CHCl₃ (100 mL),washed 1% AcOH (3×50 mL), H₂O (100 mL) dried over MgSO₄ and evaporated.The residue was purified by preparative TLC on four C18 reverse-phaseplates using 50% aqueous 2-PrOH with 0.2% TFA as eluant to give Compound76, 25 mg (22%) as an orange solid.

Example 74 Avidin-labeling with a Biotinylated Fluorescent Crown Ether

A 5 mg/mL solution of streptavidin in phosphate-buffered saline (PBS, pH7.0) is treated with a 1 mg/mL solution of biotinylated indicator (e.g.,Compound 212, Example 73) in 2% DMSO/PBS at a molar ratio such that twoequivalents of biotinylated indicator are present for every streptavidinmolecule. The resulting solution is incubated at 37° C. for 4 hours andthen centrifuged. The supernatant is applied to a Sephadex G-25 gelfiltration column (2 mL bed volume/mg protein) and eluted with PBS. Thestreptavidin-indicator complex elutes first. Fractions are analyzed byTLC to ensure that no free indicator is present in the complex. Pureproduct fractions are pooled and lyophilized. The complex is useful as abridging method to apply the indicator to any biotinylated surface orsubstance.

Example 75 Preparation of 1-Aza-benzo-15-crown-5 ether (82)

To a solution of 2-aminophenol 81 (1.322 g, 12 mmol) in MeCN (1 L),powdered CsF (7.300 g, 48 mmol) is added. The mixture is stirredvigorously for 1 h and then tetraethyleneglycole ditosylate (6.100 g, 12mmol) in MeCN (50 mL) is introduced. The mixture is refluxed under N₂atmosphere for 24 h and evaporated. The residue is dissolved in CHCl₃(800 mL), washed with H₂O, sat. NaHCO₃, H₂O, sat. NaCl (200 mL each),filtered through paper, and evaporated. The crude product is purified bycolumn chromatography on silica gel (12×60 cm bed column, made in CHCl₃)using CHCl₃ as eluant to give compound 82, 1.995 g (62% yield) as a lowmelting solid.

Example 76 Preparation of1-Methoxycarbonylmethyl-1-aza-benzo-15-crown-5-ether (83)

The mixture of compound 82 (1.380 g, 5.17 mmol), DIEA (2.4 mL, 25.84mmol), methyl bromoacetate (1.8 mL, 10.34 mmol), and Nal (0.750 g, 5.00mmol) in MeCN (100 mL) is refluxed under N₂ atmosphere for 24 h, thencooled and evaporated. The residue is re-dissolved in CHCl₃ (400 mL),washed with 1% AcOH (2×200 mL), H₂O (200 mL). The chloroform solution isdried over MgSO₄, filtered through paper, and evaporated. The crudeproduct is purified by column chromatography on silica gel (3×30 cm bedcolumn, made in CHCl₃) using 0-2.5% MeOH gradient in CHCl₃ as eluant togive compound 83, 1.102 g (63% yield) as a yellow oil.

Example 77 Preparation of15-Formyl-1-methoxycarbonylmethyl-1-aza-benzo-15-crown-5-ether (84)

To a solution of the Vilsmeier reagent prepared from POCd₃ (1.0 mL, 11.0mmol) in 5 mL DMF, compound 83 (0.750 g, 2.20 mmol) in DMF (2 mL) isintroduced. The mixture is stirred under a N₂ atmosphere for 16 h, thenpoured into ice (20 g)/sat. K₂CO₃ (50 mL) mixture. The mixture isextracted with CHCl₃ (50+5×10 mL), and the extract is dried over MagSO₄and evaporated. The crude product is purified by column chromatographyon silica gel (1.5×30 cm bed column, made in CHCl₃) using CHCl₃ aseluant to give aldehyde 84, 0.632 g (78% yield) as an off-whitelow-melting solid.

Example 78 Preparation of Compound 85 with a Fluorinated Xanthene as aDYE

A mixture of the aldehyde 84 (0.212 g, 0.58 mmol) and 4fluororesorcinol(0.163 g, 1.27 mmol) in methanesulfonic acid (7 mL) is stirred for 24 h,then poured into 3N NaOAc (100 mL). The precipitated solid is filteredoff, washed generously with water, and dried under vacuum to givecompound 85 (0.229 g, 67%) as an off-white solid. Crude compound 85 isused in the next step without purification.

Example 79 Preparation of a Cell-impermeable Metal Chelating Compound(Compound 86) with a Fluorinated Xanthene as a DYE

A mixture of the dihydro compound 85 (0.117 g, 0.20 mmol) and freshlypowdered chloranil (0.246 g, 1.00 mmol) in CHCl₃/MeOH 1:1 (10 mL) isvigorously stirred upon reflux for 3 h, then cooled down, filtered fromthe excess oxidizer and evaporated. The residue is purified bypreparative TLC on silica gel, using MeOH/AcOH 5%:2% in CHCl₃ as eluantto give the compound 86 (0.046 g, 39%) as an orange solid.

Example 80 Preparation of a Cell-permeable Metal Chelating Compound(Compound 87) with a Fluorinated Xanthene as a DYE

To a solution of compound 86 (12 mg, 0.02 mmol) and DIEA (75 μL, 0.4mmol) in DMF (1 mL), bromomethyl acetate (20 μL, 0.2 mmol) is added. Themixture is stirred for 3 h and evaporated at 1 mm Hg vacuum. The residueis purified by preparative TLC on silica gel, using MeOH/AcOH 7%:1% inCHCl₃ as eluant to give the compound 87 (12 mg, 96%) as an orange solid.

Example 81 Preparation of Metal Chelating Compound (Compound 89) withTetramethylrosamine as DYE

A mixture of aldehyde 844 (0.410 g, 1.11 mmol), and3-(N,N-dimethylamino)phenol (0.337 g, 2.46 mmol), and TsOH (22 mg) inEtCOOH (11 mL) is stirred for 16 h at 65° C. The mixture is cooled downand poured into 3N NaOAc (100 mL). The resulting suspension is extractedwith CHCl₃ (100+7×20 mL). The chloroform extract is filtered throughpaper and evaporated. The crude dihydro compound 88 is re-dissolved inMeOH/CHCl₃ 1:1 mixture (10 mL) and treated with chloranil (0.246 g, 1.00mmol). The oxidation is continued upon vigorous stirring for 2 h, thenthe solvents are evaporated, the residue is re-dissolved in CHCl₃, andpurified by column chromatography on silica gel (4×60 cm bed column,made with 5% MeOH+1% AcOH in CHCl₃) using (5-12.5%) MeOH/(1.0-1.3%) AcOHgradient as eluant to give the crude product, which is purified furtherby preparative TLC on silica gel, using 12% MeOH/3% AcOH in CHCl₃ aseluant to give compound 89 (0.038 g, 6%) as a dark red solid.

Example 82 Preparation of a Metal Chelating Compound (Compound 90) witha Borapolyazaindacene as a DYE

To a stirred solution of aldehyde 84 (0.367 g, 1.0 mmol) in CH₂Cl₂ (40ml) 2,4-dimethylpyrrole (0.25 mL, 2.4 mmol) is added, followed by TFA(0.09 mL, 1.2 mmol). The mixture is stirred for 20 h, diluted with CHCl(200 mL) and washed with 2% Me₄NOH (2×200 mL), H₂O (200 mL). Tyechloroform layer is separated, filtered through paper filter andevaporated. The residue is co-evaporated with toluene (50 mL),re-dissolved in toluene (40 mL) and stirred 2 h with chloranil (0.296 g,1.2 mmol). DIEA (1.7 mL, 10 mmol) is added, followed by BF3OEt₂ (1.04mL, 8 mmol) and the mixture is stirred for 20 h, filtered throughCellite, and evaporated. The residue is purified by columnchromatography on silica gel (4×40 cm bed, made in 2% MeOH+1% AcOH inCHCl₃), using the same mixture as eluant to give compound 90 (0.169 g,30%) as an orange solid.

Example 83 Synthesis of an α-nitrostilbene (Compound 92)

A mixture of aldehyde 84 (0.367 g, 1.0 mmol), Wittig salt 91 (0.804 g,1.5 mmol), and K₂CO₃ (0.690 g, 5.0 mmol) in DMF (6 mL) is stirred for 7h at 90° C., then left overnight at rt. The mixture is poured into H₂O(200 mL), and the resulting suspension is extracted with CHCl₃(200+10×20 mL). The extract is evaporated to dryness at 3 mm Hg and theresidue is purified by column chromatography on silica gel (4×60 cm bedcolumn, prepared in 4% MeOH in CHCl₃) using the same mixture of solventsas eluant to give styrene 92 (0.530 g, 97%) as a dark red low-meltingsolid.

Example 84 Preparation of a Metal Chelating Compound (Compound 93) withIndole as a DYE

A solution of silylene 92 (0.530 g, 0.97 mmol) in P(OEt)₃ (5 mL) isheated at 125° C. for 4 h, then evaporated. The residue is purified bycolumn chromatography on silica gel (4×50 cm bed column, prepared in 5%MeOH in CHCl₃) using the same mixture of solvents as eluant to givecompound 93 (0.171 g, 34%).

Example 85 Preparation of15-Nitro-1-methoxycarbonylmethyl-1-aza-benzo-15-crown-5-ether (Compound94)

To a stirred solution of crown ether 83 (0.420 g, 1.24 mmol) in Ac₂O (10mL), 65% HNO₃ (0.10 mL, 1.5 mmol) is introduced at 0° C. The mixture isstirred for 2 h at 0° C. and then poured into sat. K₂CO₃ (100 mL), andstirred for 1 h. The resulting solution is extracted with CHCl₃(100+7×20 mL), the extract is filtered through paper and evaporated. Theresidue is purified by column chromatography on silica gel (4×60 cm bedcolumn, prepared in CHCl₃) using CHCl₃ as eluant to give nitroderivative 94 (0.272 g, 57%) as a yellow-orange solid.

Example 86 Preparation of Azo-dye Metal Chelating Compound (Compound 96)

A solution of crown ether 83 (0.339 g, 1.0 mmol) in dioxane (1 mL) andAcOH (1 mL) is treated with a solution of diazo compound 95, preparedfrom sulfanilic acid (0.190 g, 1.1 mmol) in 6 mL H₂O. The mixture isstirred for 3 h, poured into H₂O (100 mL) and extracted with CHCl₃(100+25×20 mL). The chloroform extract is evaporated, and the residue ispurified by column chromatography on Sephadex LH-20 (3×35 cm bed column,prepared in H₂O) using H₂O as eluant to give azo compound 96 (0.029 g,7%) as a dark red solid.

Example 87 Preparation of15-Amino-1-methoxycarbonylmethyl-1-aza-benzo-15-crown-5-ether (Compound97)

A) A sample of nitro compound 94 (0.220 g, 0.57 mmol) is hydrogenated at50 psi in DMF (25 mL) over 10% Pc/C (40 mg, catalyst) for 7 h. Themixture is filtered, and evaporated at 1 mm Hg. The residue is purifiedby preparative TLC on silica gel, using 20% MeOH in CHCl₃ as eluant togive the amine 97 (128 mg, 63%).

B) A sample of azo compound 96 (4 mg, 0.01 mmoL) in 0.5 ml ethanol istreated with sodium dithionite (10 mg, 0.06 mmol) in 0.2 ml water. Thesolvents are evaporated and the residue is washed with water to giveamine 97 (3 mg, 85%), identical to that prepared in the above example.

Example 88 Preparation of Cell-permeable Compound 98

To a stirred solution of the amine 97 (25 mg, 0.07 mmol) and DIEA (0.17ml, 1 mmol) in CH₂Cl₂ (3 mL) an acid chloride, prepared from fluorinatedxanthene acid (55 mg, 0.11 mmol) in CH₂Cl₂ (2 mL) is added dropwise. Themixture is stirred for 2 h and evaporated. The residue is purified bypreparative TLC on silica gel, using 5% MeOH and 1% AcOH in CHCl₃ aseluant to give the compound 98 (32 mg, 63%) as an off-white solid.

Example 89 Preparation of Cell-impermeable Compound 99

To a solution of the compound 98 (10 mg, 0.012 mmol) in MeOH (2 mL) anaqueous solution of NH₃ (0.5 mL) is added. The mixture is stirred for 30min, evaporated to dryness, and the residue is suspended in water (2mL), and centrifuged. The supernatant is discarded, and the solid ispurified with preparative TLC on reverse-phase C18 plates, using 50%2-PrOH and 0.2% TFA in H₂O as eluant to give compound 99 (4 mg, 48%), asan orange solid.

Example 90 Preparation of 1-Aza-benzo-14-crown-4 ether (Compound 100)

To a solution of 2-aminophenol 81 (1.322 g, 12 mmol) in MeCN (1 L),powdered CsF (7.300 g, 48 mmol) is added. The mixture is stirredvigorously for 1 h and then triethyleneglycol ditosylate (5.503 g, 12mmol) in MeCN (50 mL) is introduced. The mixture is refluxed under N₂atmosphere for 16 h and then evaporated. The residue is dissolved inCHCl₃ (500 mL), washed with H₂O, sat. NaHCO₃, H₂O, sat. NaCl (200 mLeach), filtered trough paper, and evaporated. The residue is purified bycolumn chromatography on silica gel (12×60 cm bed column, made in CHCl₃)using CHCl₃ as eluant to give crude product, which is purified by columnchromatography on silica gel (12×60 cm bed column, made in 25% EtOAc inhexanes) using the same mixture of solvents as eluant to give compound100, 0.370 g (14% yield) as an off-white solid.

Example 91 Preparation of1-Methoxycarbonylmethyl-1-aza-benzo-12-crown-4-ether (Compound 101)

The mixture of compound 100 (0.360 g, 1.61 mmol), DIEA (0.56 mL, 3.22mmol), methyl bromoacetate (0.76 mL, 8.05 mmol), and Nal (0.241 g, 1.61mmol; catalyst) in MeCN (30 mL) is refluxed under N₂ atmosphere for 16h, then cooled down and evaporated. The residue is redissolved in CHCl₃(200 mL), washed with 1% AcOH (2×200 mL), H₂O (200 mL). The chloroformsolution is dried over MgSO₄, filtered through paper, and evaporated.The residue (TLC-pure crude product) is used in the next step withoutpurification, considering it to be a 1.6 mmol quantity.

Example 92 Preparation of13-Formyl-1-methoxycarbonylmethyl-1-aza-benzo-12-crown-4-ether (Compound102)

To a solution of the Vilsmeler reagent prepared from POCl₃ (0.75 mL, 8.0mmol) in 8 mL DMF compound 101 (1.60 mmol) in DMF (2 mL) is introduced.The mixture is stirred under a N₂ atmosphere for 16 h, then poured intoice (20 g)/sat. K₂CO₃ (50 mL) mixture. The mixture is extracted withCHCl₃ (100+6×10 mL); the extract is dried over MagSO₄, and evaporated.The crude product is purified by column chromatography on silica gel(1.5×30 cm bed column, made in CHCl₃) using CHCl₃ as eluant to givealdehyde 102, 0.285 g (55% yield on two steps) as an off-white solid.

Example 93 Preparation of Compound 103

A mixture of the aldehyde 102 (0.272 g, 0.88 mmol) and 4-fluororesorcinol (0.248 g, 1.94 mmol) in MsOH (13 mL) is stirred for 24 h andthen poured into 3N NaOAc (100 mL). The mixture is centrifuged, and thesupernatant is discarded. The solid is washed with water, and dried invacuum to give compound 103 (0.087 g, 18%) as an off-white solid. Crudecompound 104 is used in the next step without purification.

Example 94 Preparation of Cell-impermeable Compound 104

A mixture of the dihydro compound 85 (0.085 g, 0.16 mmol), and freshlypowdered chloranil (0.197 g, 0.80 mmol) in CHCl_(3/)MeOH 1:1 mixture (6mL) is vigorously stirred during reflux for 4 h, then cooled down,filtered from the excess oxidizer and evaporated. The residue ispurified by preparative TLC on silica gel, using MeOH/AcOH 5%:2% inCHCl₃ as eluant to give the compound 104 (0.031 g, 36%) as an orangesolid.

Example 95 Preparation of 1-Aza-benzo-18-crown-6 ether (Compound 105)

To a solution of 2-aminophenol 81 (1.090 g, 10 mmol) in MeCN (1 L),powdered CsF (6.080 g, 40 mmol) is added. The mixture is stirredvigorously for 1 h and then pentaethyleneglycol ditosylate (5.500 g, 11mmol) in MeCN (50 mL) is introduced. The mixture is refluxed under N₂atmosphere for 70 h and then evaporated. The residue is dissolved inCHCl₃ (600 mL), washed with H₂O, sat. NaHCO₃, H₂O, and sat. NaCl (200 mLeach). The chloroform solution is dried over MgSO₄, filtered troughpaper, and evaporated. The residue is purified by column chromatographyon silica gel (8×45 cm bed column, made in CHCl₃) using 0-1.2% MeOHgradient in CHCl₃ as eluant to give compound 105, 0.681 g (22% yield) asa off-white solid.

Example 96 Preparation of1-Methoxycarbonylmethyl-1-aza-benzo-18-crown-6-ether (Compound 126)

A mixture of compound 105 (0.670 g, 2.15 mmol), DIEA (0.72 mL, 4.15mmol), methyl bromoacetate (1.01 mL, 10.75 mmol), and Nal (0.322 g, 2.15mmol; catalyst) in MeCN (40 mL) is refluxed under a N₂ atmosphere for 16h, then cooled down and evaporated. The residue is redissolved in CHCl₃(200 mL), washed with 1% AcOH (2×200 mL), H₂O (200 mL). Chloroformsolution is dried over MgSO₄, filtered trough paper, and evaporated. Theresidue is purified by column chromatography on silica gel (3×30 cm bedcolumn, made in CHCl₃) using 2.5-4% MeOH gradient in CHCl₃ as eluant togive compound 106, 0.430 g (52% yield) as an off-white solid.

Example 97 Preparation of18-Formyl-1-methoxycarbonylmethyl-1-aza-benzo-18-crown-6-ether (Compound107)

To a solution of the Vilsmeier reagent prepared from POCl₃ (1.23 mL,13.2 mmol) in 6 mL DMF compound 106 (0.410 g, 1.32 mmol) in DMF (2 mL)is introduced. The mixture is stirred under a N₂ atmosphere for 16 h,then poured into ice (40 g)/sat. K₂CO₃ (100 mL) mixture. The mixture isextracted with CHCl₃ (5×100 mL), the extract is dried over MagSO₄, andevaporated. The crude product is treated with cold ether (10 mL), andthe precipitated solid is collected to give aldehyde 107, 0.246 g (45%yield) as a white solid.

Example 98 Preparation of Compound 108

A mixture of the aldehyde 107 (0.236 g, 0.57 mmol) and 4-fluororesorcinol (0.186 g, 1.45 mmol) in MsOH (8 mL) is stirred for 16 h, thenpoured into 3N NaOAc (100 mL). The precipitate is filtered, washed withwater, and dried in vacuum to give compound 108 (0.350 g, 97%) as anoff-white solid. Crude compound 108 is used in the next step withoutpurification.

Example 99 Preparation of Cell-impermeable Compound 109

A mixture of the dihydro compound 108 (0.350 g, 0.56 mmol), and freshlypowdered chloranil (0.701 g, 2.85 mmol) in CHCl_(3/)MeOH 1:1 mixture (25mL) is vigorously stirred upon reflux for 4 h, then cooled down,filtered from the excess oxidizer and evaporated. The residue ispurified by column chromatography on silica gel, (4×40 cm bed column,made in 10% MeOH+1% AcOH in CHCl₃) using the same mixture of solvents aseluant to give the compound 109 (0.088 g, 25%) as an orange solid.

Example 100 Preparation of Cell-permeable Compound 110

To a solution of compound 109 (32 mg, 0.05 mmol) and DIEA (0.17 mL, 1.0mmol) in DMF (2 mL), bromomethyl acetate (50 □L, 0.5 mmol) is added. Themixture is stirred for 3 h and evaporated at 1 mm Hg vacuum. The residueis purified by preparative TLC on silica gel, using MeOH/AcOH 15%:1% inCHCl₃ as eluant to give the compound 110 (20 mg, 57%) as an orangesolid.

Example 101 Preparation of 1-(2′-Methoxyacetyl)-1-aza-benzo-15crown-5ether (Compound 111)

To a stirred solution of the crown ether 82 (6.20 g, 23 mmol) and Et₃N(9.3 mL, 70 mmol) in CH₂Cl₂ (250 mL) a methoxyacetyl chloride (3.2 mL,35 mml) in CH₂Cl₂ (15 mL) is introduced within 15 min. The mixture isstirred for 3 h, diluted with CHCl₃ (150 mL), and washed with H₂O, 1%AcOH, H₂O, sat. NaHCO₃, H₂O (200 mL each), filtered through paper filterand evaporated. The residue is purified by column chromatography onsilica gel (6×45 cm bed column, made in CHCl₃), using 2.5% MeOH in CHCl₃as eluant to give compound 111 (4.25 g, 55%) as a yellow oil.

Example 102 Preparation of 1-(2′-Methoxyethyl)-1-aza-benzo-15-crown-5ether (Compound 112)

A) To a stirred solution of compound 111 (4.25 g, 12.5 mmol) in dry THF(50 mL) a 1N diborane in THF (50 mL, 50 mmol) is added. The mixture isstirred under reflux for 20 h, decomposed by dropwise addition of MeOH(50 mL), and evaporated. The residue is co-evaporated with MeOH (5×100mL) to remove boron complexes, and dried in vacuo to give compound 112(4.05 g, 99%) as a colorless oil.

B) A mixture of compound 82 (1.20 g, 4.50 mmol), DEIA (1.6 mL, 9.0mmol), 2-bromoethylmethyl ether (4.2 mL, 45 mmol), Nal (0.68 g, 4.50mmo, catalyst) in MeCN (200 mL) is stirred under reflux for 70 h. MoreDIEA (1.6 mL, 9.0 mmol) and 2-bromoethylmethyl ether (4.2 mL, 45 mmol)are added and heating continued for another 40 h. The reaction mixtureis evaporated, the residue is re-dissolved in CHCl₃ (300 mL), washedwith 1% AcOH (3×200 mL), H₂O (200 mL), filtered through paper filter andevaporated. The residue is purified by column chromatography on silicagel (3×40 cm bed column, made in 2.5% MeOH in CHCl₃), using the samemixture of solvents as eluant to give compound 112 (0.080 g, 5%),identical to that prepared section A of this example.

Example 103 Preparation of1-(2′-Methoxyethyl)-15-formyl-1-aza-benzo-15-crown-5 ether (Compound113)

To a solution of the Vilsmeier reagent prepared from POCl₃ (12 mL, 125mmol) in 70 mL DMF compound 112 (4.05 g, 12.5 mmol) in DMF (10 mL) isintroduced. The mixture is stirred under N₂ atmosphere for 16 h at 40°C., then more Vilsmeier reagent from POCl₃ (12 mL, 125 mmol) in 70 mLDMF is added and the stirring continued for another 24 h. The mixture ispoured into ice (400 g)/sat. K₂CO₃ (400 mL) mixture. The mixture isextracted with CHCl₃ (300+6×50 mL), the extract is dried over MagSO₄,and evaporated. The crude product is purified by column chromatographyon silica gel (6×50 cm bed column, made in CHCl₃) using 1.5% MeOH inCHCl₃ as eluant to give aldehyde 113, 1.613 g (36% or, 58% based onrecovery), then eluant is changed to 10% to recover starting material(1.49 g, 37%).

Example 104 Preparation of Compound 114

A mixture of the aldehyde 113 (0.700 g, 1.98 mmol) and4-fluororesorcinol (0.558 g, 4.36 mmol) in methanesulfonic acid (30 mL)is stirred for 24 h and poured into 3N NaOAc (300 mL). The mixture isextracted with n-BuOH (7×50 mL). The extract is evaporated to give thecrude dihydro derivative 114. Crude compound 114 is used in the nextstep without purification, considering it as 1.8 mmol.

Example 105 Preparation of Cell-impermeable Compound 115

A mixture of the dihydro compound 114 (1.8 mmol), and freshly powderedchloranil (2.210 g, 9.00 mmol) in CHCl_(3/)MeOH 1:1 mixture (100 mL) isvigorously stirred upon reflux for 4 h, then cooled down, filtered fromthe excess oxidizer and evaporated. The residue is purified by columnchromatography on silica gel (6×55 cm bed column, made in 10% MeOH+1%AcOH in CHCl₃), using 10-15% MeOH gradient in CHCl₃+1% AcOH as eluant togive the compound 115 (0.189 g, 18% for two steps) as a brown solid.

Example 106 Preparation of Cell-permeable Compound 116

To a solution of compound 115 (28 mg, 0.05 mmol) and DIEA (0.17, 1.0mmol) in DMF (2 mL), bromomethyl acetate (50 μL, 0.5 mmol) is added. Themixture is stirred for 1 h and evaporated at 1 mm Hg vacuum. The residueis purified by preparative TLC on silica gel, using MeOH/AcOH 10%:2% inCHCl₃ as eluant to give the compound 116 (4 mg, 12%) as an orange solid.

Example 107 Preparation of Tetramethylrosamine Compound 118

A mixture of aldehyde 113 (0.900 g, 2.55 mmol), and3-(N,N-dimethylamino)phenol (0.769 g, 5.61 mmol), and TsOH (50 mg,catalyst) in EtCOOH (25 mL) is stirred for 16 h at 65° C. The mixture iscooled down and poured into 3N NaOAc (500 mL). The resulting suspensionis extracted with CHCl₃ (200+7×30 mL). The extract is filtered throughpaper and evaporated. The resulting crude dihydro compound 117 isre-dissolved in MeOH/CHCl₃ 1:1 mixture (100 mL) and treated withchloranil (0.541 g, 2.20 mmol). The oxidation is continued upon vigorousstirring for 2 h, then the solvents are evaporated, the residue isre-dissolved in CHCl₃, and purified by column chromatography on silicagel (8×50 cm bed column, made with 5% MeOH+1% AcOH in CHCl₃) using 5-20%MeOH gradient in CHCl₃+1.0% AcOH as eluant to give compound 118 (0.236g, 16% on two steps) as a dark red solid.

Example 108 Synthesis of an α-nitrostilbene (Compound 119)

A mixture of aldehyde 113 (0.172 g, 0.49 mmol), Wittig salt 91 (0.392 g,0.73 mmol), and K₂CO₃ (0.338 g, 2.45 mmol) in DMF (3 mL) is stirred for6 h at 90° C., then left overnight at rt. The mixture is poured into H₂O(200 mL), and the resulting suspension is extracted with CHCl₃ (50+7×20mL). The extract is evaporated to dryness at 3 mm Hg and the residue ispurified by column chromatography on silica gel (2.5×30 cm bed column,prepared in 3% MeOH in CHCl₃) using the same mixture of solvents aseluant to give stilbene 119 (0.195 g, 75%) as a dark red low-meltingsolid.

Example 109 Preparation of Compound 120

A solution of stilbene 119 (0.190 g, 0.36 mmol) in P(OEt)₃ (3 mL) isheated at 125° C. for 4 h, then evaporated. The residue is purified bycolumn chromatography on silica gel (2.5×50 cm bed column, prepared in5% MeOH and 1% AcOH in CHCl₃) using the same mixture of solvents aseluant to give compound 120 (0.099 g, 55%) as an off-white solid.

Example 110 Preparation of 1-Ethoxyoxalyl-1-aza-benzo-15-crown-5-ether(Compound 121)

To a mixture of compound 82 (1.345 g, 5.00 mmol) and Et₃N (1.4 mL, 10.00mmol) in CH₂Cl₂ (30 mL), ethyl oxalylchloride (0.84 mL, 7.50 mmol) inCH₂Cl₂ (5 mL) is added dropwise. The mixture is stirred for 1 h, dilutedwith CHCl₃ (200 mL), washed with 1% AcOH (3×100 mL), H₂O (100 mL), sat.NaHCO₃ (2×100 mL), sat. NaCl (200 mL). The chloroform fraction is driedover MgSO₄ and evaporated. The residue is purified by columnchromatography on silica gel (4×30 cm bed, made in 30% EtOAc inhexanes), using 30-60% EtOAc gradient in hexanes as eluant to givecompound 121 (1.170 g, 65% yield) as a yellow oil.

Example 111 Preparation of1-(N,N-Dimethylaminooxalyl)-1-aza-benzo-15-crown-5-ether (Compound 122)

To a solution of the compound 121 (0.734 g, 2.00 mmol) in MeOH (15 mL),a solution of dimethylamine (2 mL, 40 mmol) in cold MeOH (15 mL) isadded upon cooling to 0° C. The mixture is heated to 30° C. for 16 h,then cooled to 0° C. and a new portion of dimethylamine (2 mL, 40 mmol)is introduced. The mixture is stirred for 3 h at 30° C. and thenevaporated and the residue is treated with cold ether. The precipitatedsolid is filtered off, and washed with ether to give compound 122 (0.542g, 74% yield) as a white solid.

Example 112 Preparation of1-(2′-N,N-Dimethylaminoethyl)-1-aza-benzo-15-crown-5-ether (Compound123)

To the solution of compound 122 (0.440 g, 1.20 mmol) in THF (10 ml) asolution of 1N diborane in THF (12 mL, 12 mml) is added. The mixture isrefluxed for 16 h, then decomposed by careful addition of MeOH (30 mL),evaporated, and co-evaporated with MeOH (5×50 mL) to remove boroncomplexes. The crystalline residue of the compound 123 (0.405 g, 100%yield) is used in next step without purification.

Example 113 Preparation of1-(2′-N,N-Dimethylaminoethyl)-15-formyl-1-aza-benzo-15-crown-5-ether(Compound 124)

To a solution of the Vilsmeier reagent prepared from POCl₃ (1.5 mL, 16.0mmol) in 5 mL DMF, compound 123 (0.550 g, 1.62 mmol) in DMF (2 mL) isintroduced. The mixture is stirred under N₂ atmosphere for 16 h, thenpoured into ice (20 g)/sat. K₂CO₃ (50 mL) mixture. The mixture isextracted with CHCl₃ (100+7×25 mL), the extract is dried over MagSO₄,and evaporated. The crude product is purified by column chromatographyon silica gel (3×35 cm bed column, made with 3% MeOH in CHCl₃) using thesame mixture of solvents as eluant to give aldehyde 124, (0.175 g 30%yield) as a yellow low-melting solid.

Example 114 Synthesis of α-nitrostilbene (Compound 125)

A mixture of aldehyde 124 (0.070 g, 0.19 mmol), Wittig salt 91 (0.154 g,0.29 mmol), and K₂CO₃ (0.131 g, 0.95 mmol) in DMF (2 mL) is stirred for16 h at 90° C. The mixture is diluted with CHCl₃ (10 mL), filtered frominorganic materials and evaporated to dryness at 3 mm Hg. The residue ispurified by preparative TLC on silica gel using 20% MeOH and 5% AcOH inCHCl₃ as eluant to give stilbene 125 (0.060 g, 58%) as a dark red solid.

Example 115 Preparation of Compound 126

A solution of stilbene 119 (0.055 g, 0.01 mmol) in P(OEt)₃ (2 mL) isheated at 125° C. for 14 h, then evaporated. The residue is purified bypreparative TLC on silica gel using 25% MeOH and 5% AcOH in CHCl₃aseluant to give compound 126 (0.029 g, 57%) as an off-white solid.

Example 116 Hydrolysis of Compound 86

To a solution of the compound 86 (0.032 g, 0.055 mmol) in 50% MeOH (2mL), 1N KOH (0.16 mL, 16 mml) is added. The mixture is stirred for 16 h,diluted with H₂O, and 0.2 N HCl is added to achieve pH=8.0. Theresulting solution is filtered through a nylon membrane filter andevaporated. The crude product is purified by column chromatography onSephadex LH-20 (2.6×50 cm bed column, made with H₂O), using H₂O aseluant to give compound 127 (0.021 mg, 59% yield) as a brown solid.

Example 117 Preparation of Compound 138

To a solution of compound 128 in dry THF (0.5M) and two equivalents ofDIEA is added dropwise benzoyl chloride (1.0 eq). After 1 h at roomtemperature, the volatiles are removed. The residue is partitionedbetween 0.1M HCl and ethyl acetate. The organic layer is washed withbrine, dried over sodium sulfate, and concentrated to give amide 129.

To a solution of 129 in THF (0.5M) is added a solution of borane-THF(1.0M, 2 eq) at room temperature. The resulting solution is stirred atroom temperature over night and then methanol (2 eq) is carefully added.The volatiles are removed in vacuo, and the residue partitioned betweenwater and ethyl acetate. The organic layer is dried over sodium sulfateand concentrated to give aniline 130, which is purified further ifnecessary by flash chromatography on silica gel using ethyl acetate inhexanes.

To a solution of 130 (0.05M) in dry THF with 2 eq DIEA is slowly added a0.1M solution of diglycolyl chloride in dry THF with rapid stirring.After 2 h, the reaction solution is concentrated in vacuo. The residueis partitioned between 0.1M HCl and ethyl acetate. The organic layer iswashed with brine, dried over sodium sulfate, and concentrated to givebisamide 131.

To a 0.1M solution of 131 in dry THF is slowly added borane-THF (1.0M, 5eq). The resulting solution is stirred at room temperature overnight andthen methanol (5 eq) is added. The resulting mixture is concentrated invacuo, and the residue partitioned between water and ethyl acetate. Theorganic phase is washed with brine and dried over sodium sulfate, andconcentrated in vacuo to give the diol 132, which is purified further ifnecessary by flash chromatography on silica gel using methanol inchloroform.

A solution of 132 is dissolved in dry acetonitrile to 0.5M. Theresulting solution is treated with methanesulfonyl chloride (1.2 eq) andcesium carbonate (2 eq). The resulting mixture is stirred at reflux for6 hours, then cooled and filtered and concentrated in vacuo. The desiredazacrown ether 133 is formed as a minor product, isolated from theresidue by flash chromatography on silica gel using ethyl acetate inhexanes.

The crown 133 is stirred in ethyl acetate (0.5M) with 10 wt % Pd/C(cat.) under 30 psi hydrogen gas for 6 hours. After filtration, thereaction solution is concentrated in vacuo to give aniline 134.

To a solution of 134 in DMF (0.5M) is added methyl bromoacetate (5 eq)and DIEA (3 eq). The resulting solution is stirred at 90° C. overnight,then cooled and concentrated. The residue is partitioned between 0.1MHCl and ethyl acetate. The organic layer is washed with brine and driedover sodium sulfate, then concentrated in vacuo to give crown 135, whichis purified further if necessary by flash chromatography on silica gelusing ethyl acetate in hexanes.

To a solution of Vilsmeier reagent made from 2 eq phosphorousoxychloride in DMF is added a solution of 135 in DMF. The resultingsolution is stirred at room temperature overnight, then poured into aq.sodium bicarbonate. The resulting mixture is extracted with ethylacetate. The extract is washed with water and brine, dried over sodiumsulfate, and concentrated in vacuo to give aldehyde 136, which ispurified further if necessary by crystallization from methanol.

To a solution of 136 in methanesulfonic acid (0.5M) is added4fluororesorcinol (2 eq). The resulting solution is stirred at roomtemperature 15 minutes, then poured carefully into 3M aqueous NaOAc. Theresulting precipitate is collected by suction filtration, rinsed withwater, and dried in vacuo to give dihydro compound 137 as a pale brownpowder.

A solution of 137 in methanol/chloroform (1:1, 0.5M) is treated with 2eq of para-chloranil. The resulting mixture is stirred at roomtemperature overnight, then filtered and concentrated. The residue ispurified by chromatography on silica gel using methanol in chloroform togive indicator 138 as a dark orange powder.

Example 118 Synthesis of Compound 140

A 0.5M solution of compound 86 in 1:1 methanol/dioxane is treated atroom temperature with 5 equivalents of tetrabutylammonium hydroxide.After 2 hours the volatiles are removed in vacuo and the residue isdissolved in water. The pH is lowered to 2.0 by dropwise addition ofaqueous HCl. The resulting precipitate is collected by filtration anddried in vacuo to give carboxylic acid 139. A 0.5M solution of compound139 in dry DMF is treated with 1.5 equivalents ofO-(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate. After2 hours the reaction soluted is diluted 10× with diethyl ether. Theresulting precipitate is collected by filtration to give reactivesuccinimidyl ester compound 140.

Example 119 Synthesis of Compound 141

A 0.1M solution of compound 140 in DMSO is added to a 0.1M solution ofan equivalent mass of aminodextran (average MW 10000) in 0.3M sodiumbicarbonate solution. The resulting solution is stirred at rt overnight,then slowly diluted with 10× methanol. The resulting precipitate iscollected by centrifugation to give conjugated substance 141, which canbe purified further if necessary by gel filtration chromatography usingwater and Sephadex G-15.

All publications, patents and patent applications referred to withinthis document are incorporated by reference to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by reference.

The reagents employed in the examples are commercially available or canbe prepared using commercially available instrumentation, methods, orreagents known in the art. The foregoing examples illustrate variousaspects of the invention and practice of the methods of the invention.The examples are not intended to provide an exhaustive description ofthe many different embodiments of the invention. Thus, although theforgoing invention has been described in some detail by way ofillustration and example for purposes of clarity of understanding, thoseof ordinary skill in the art will realize readily that many changes andmodifications can be made thereto without departing from the spirit orscope of the appended claims.

1. A crown ether chelating compound having the formula:

wherein R¹ is selected from the group consisting of -L-R_(x), -L-S_(c),-L-DYE, C₁-C₁₈ alkyl and C₇-C₁₈ arylalkyl, each of which is optionallysubstituted by halogen, azido, nitron, nitroso, amino, hydroxy, cyano,C₁-C₆ alkoxy, an aryl or heteroaryl ring system, —(SO₂)—R¹⁵,—(SO₂)—O—R¹⁵, —(C═O)—R¹⁵, —(C═O)—O—R¹⁶, —(C═O)—NR¹⁷R¹⁸, C₁-C₆alkylamino, C₂-C₁₂ dialkylamino, C₁-C₆ alkyl or C₁-C₆ alkoxy, each ofwhich is itself optionally substituted by halogen, amino (—NR¹⁷R¹⁸),hydroxy, —(SO₂)—R¹⁵, —(SO₂)—O—R¹⁵, —(C═O)—R¹⁵, —(C═O)—O—R¹⁶ or—(C═O)—NR¹⁷R¹⁸; each L is independently a covalent linkage; each R_(x)is independently a radical of a reactive group selected from the groupconsisting of an acrylamide, an activated ester of a carboxylic acid, acarboxylic ester, an acyl azide, an acyl nitrile, an aldehyde, an alkylhalide, an anhydride, an aniline, an amine, an aryl halide, an azide, anaziridine, a boronate, a diazoalkane, a haloacetamide, a halotriazine, ahydrazine, an imido ester, an isocyanate, an isothiocyanate, amaleimide, a phosphoramidite, a reactive platinum complex, a silylhalide, a sulfonyl halide, a thiol and a photoactivatable group; eachS_(c) is independently a radical of a conjugated substance selected fromthe group consisting of an amino acid, a peptide, a protein, apolysaccharide, a nucleoside, a nucleotide, an oligonucleotide, anucleic acid, a hapten, a psoralen, a drug, a hormone, a lipid, a lipidassembly, a synthetic polymer, a polymeric microparticle, a biologicalcell, a virus, an antibody or fragment thereof, an avidin orstreptavidin, a biotin, a blood component protein, a dextran, an enzyme,an enzyme inhibitor, a hormone, an IgG binding protein, a fluorescentprotein, a growth factor, a lectin, a lipopolysaccharide, amicroorganism, a metal binding protein, a metal chelating moiety, anon-biological microparticle, a peptide toxin, aphosphotidylserine-binding protein, a structural protein, asmall-molecule drug, or a tyramide; each DYE is independently a radicalof a reporter molecule selected from the group consisting of xanthene,borapolyazaindacene, carbocyanine, benzofuran, quinazolinone, indole, abenzazole, oxazine, and coumarin able to form a covalent bond; R⁷, R⁸and R¹⁰ are independently selected from the group consisting of H,halogen, azido, nitro, nitroso, amino, cyano, -L-R_(x), -L-S_(c),-L-DYE, C₁-C₆ alkyl or C₁-C₆ alkoxy, each of which is itself optionallysubstituted by halogen, amino, hydroxy, —(SO₂)—R¹⁵, —(SO₂)—O—R¹⁵,—(C═O)—R¹⁵, —(C═O)—O—R¹⁶, or —(C═O)—NR¹⁷R¹⁸; R¹⁵ is selected from thegroup consisting of H, C₁-C₆ alkyl, -L-R_(x), -L-S_(c) and -L-DYE; R¹⁶is selected from the group consisting of H, C₁-C₆ alkyl, benzyl, abiologically compatible esterifying group, a biologically compatiblesalt, -L-R_(x), -L-S_(c) and -L-DYE; R¹⁷ and R¹⁸ are independentlyselected from the group consisting of H, C₁-C₆ alkyl, C₁-C₆carboxyalkyl, alpha-acyloxyalkyl, trialkylsilyl a biologicallycompatible salt, -L-R_(x), -L-S_(c) and -L-DYE; or R¹⁷ and R¹⁸ taken incombination form a 5- or 6-membered aliphatic ring that optionallyincorporates an oxygen atom; R¹⁹ and R²⁰ are independently selected fromthe group consisting of H, halogen, azido, nitro, nitroso, amino, cyano,-L-R_(x), -L-S_(c), -L-DYE, C₁-C₆ alkyl and C₁-C₆ alkoxy, each of whichis itself optionally substituted by halogen, amino, hydroxy, —(SO₂)—R¹⁵,—(SO₂)—O—R¹⁵, —(C═O)—R¹⁵, —(C═O)—O—R¹⁶, or —(C═O)—NR¹⁷R¹⁸; or R¹⁹ andR²⁰ taken in combination from a fused six-membered benzo moiety that isoptionally substituted by halogen, azido, nitro, nitroso, amino, cyano,-L-R_(x), -L-S_(c), -L-DYE, C₁-C₆ alkyl or C₁-C₆ alkoxy, each of whichis itself optionally substituted by halogen, amino, hydroxy, —(SO₂)—R¹⁵,—(SO₂)—O—R¹⁵, —(C═O)—R¹⁵, —(C═O)—O—R¹⁶, or —(C═O)—NR¹⁷R¹⁸; or adjacentsubstituents R⁷—R⁸, taken in combination, form a fused six-memberedbenzo moiety, which is optionally substituted by halogen, azido, nitro,nitroso, amino, cyano, -L-R_(x), -L-S_(c), -L-DYE, C₁-C₆ alkyl or C₁-C₆alkoxy, each of which is optionally substituted by halogen, amino,hydroxy, —(C═O)—R¹⁵, —(C═O)—O—R¹⁶, or —(C═O)—NR¹⁷R¹⁸; or any twoadjacent substituents R⁷—R⁸, or R¹⁹ and R²⁰, taken in combination witheach other, form a fused DYE.
 2. The compound according to claim 1,wherein said —R_(x) is a radical of a reactive group selected from thegroup consisting of carboxylic acid, succinimidyl ester of a carboxylicacid, hydrazide, amine and a maleimide.
 3. The compound according toclaim 1, wherein said —S_(c) is a radical of a conjugated substanceselected from the group consisting of an amino acid, a peptide, aprotein, a polysaccharide, a nucleoside, a nucleotide, anoligonucleotide, a nucleic acid, a hapten, a psoralen, a drug, ahormone, a lipid, a lipid assembly, a synthetic polymer, a polymericmicroparticle, a biological cell and a virus.
 4. The compound accordingto claim 1, wherein said —S_(c) is a radical of a conjugated substanceselected from the group consisting of an antibody or fragment thereof,an avidin or streptavidin, a biotin, a blood component protein, adextran, an enzyme, an enzyme inhibitor, a hormone, an IgG bindingprotein, a fluorescent protein, a growth factor, a lectin, alipopolysaccharide, a microorganism, a metal binding protein, a metalchelating moiety, a non-biological microparticle, a peptide toxin, aphosphotidylserine-binding protein, a structural protein, asmall-molecule drug, and a tyramide.
 5. The compound according to claim1, wherein said -DYE moiety is independently substituted by a lipophilicgroup.
 6. The compound according to claim 5, wherein said lipophilicgroup is an AM or acetate ester.
 7. The compound according to claim 1,wherein said DYE is a radical of xanthene.
 8. The compound according toclaim 7, wherein said DYE moiety is independently substituted by alipophilic group.
 9. The compound according to claim 8, wherein saidlipophilic group is an AM or acetate ester.
 10. The compound accordingto claim 1, wherein R⁷, R⁸, R¹⁰, R¹⁹ and R²⁰, when present, are H. 11.The compound according to claim 1, wherein R¹ is C₁-C₆ alkyl that issubstituted one or more times by amino (—NR¹⁷R¹⁸), —(C═O)—O—R¹⁶ or—(C═O)—NR¹⁷R¹⁸.
 12. The compound according to claim 11, wherein said R¹is methyl or ethyl.
 13. The compound according to claim 11 wherein saidR¹⁶ is selected from the group consisting of H, C₁-C₆ alkyl, benzyl, abiologically compatible esterifying group, and a biologically compatiblesalt.
 14. The compound according to claim 13 wherein said R¹⁶ is methyl.15. The compound according to claim 11 wherein said R¹⁷ and R¹⁸ are eachmethyl.
 16. The compound according to claim 1, wherein said R¹⁹ and R²⁰are H.
 17. A composition comprising: a. a compound according to claim 1;and, b. a metal ion that is capable of being chelated by said compound.18. The composition according to claim 17, wherein said metal ion isselected from the group consisting of Li⁺ and K⁺.
 19. A method forbinding a target metal ion in a sample, comprising steps of: a.contacting said sample with a metal chelating compound of claim 1; b.incubating said sample and said metal chelating compound for sufficienttime to allow said compound to chelate said target metal ion wherebysaid metal ion is bound.
 20. A method for binding and detecting targetions in a live cell, said method comprising: a) contacting a sample oflive cells with a crown ether compound of claim 1 with the proviso thatsaid compound comprise a DYE moiety and at least one lipohilic group; b)incubating said sample and said crown ether chelate compound forsufficient time to allow said compound to chelate said target metal ion;and, c) illuminating said sample with an appropriate wavelength wherebysaid target ion is detected in a live cell.